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Endocrinology Vol. 148, No. 12 5811-5821
Copyright © 2007 by The Endocrine Society

Evidence for Circadian Regulation of Activating Transcription Factor 5 But Not Tyrosine Hydroxylase by the Chromaffin Cell Clock

Dario R. Lemos, Leela Goodspeed, Luciana Tonelli, Marina P. Antoch, Sergio R. Ojeda and Henryk F. Urbanski

Division of Neuroscience (D.R.L., L.G., S.R.O., H.F.U.), Oregon National Primate Research Center, Beaverton, Oregon 97006; Department of Physiology (D.R.L.), Faculty of Medicine, University of Buenos Aires, 1121 Buenos Aires, Argentina; Vaccine and Gene Therapy Institute (L.T.), Oregon Health and Science University, Beaverton, Oregon 97006; Department of Cancer Biology (M.P.A.), Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44109; and Departments of Physiology and Pharmacology and Behavioral Neuroscience (H.F.U.), Oregon Health and Science University, Portland, Oregon 97239

Address all correspondence and requests for reprints to: Henryk F. Urbanski, Division of Neuroscience, Oregon National Primate Research Center, 505 Northwest 185th Avenue, Beaverton, Oregon 97006. E-mail: urbanski{at}ohsu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In mammals, adrenal medulla chromaffin cells constitute a fundamental component of the sympathetic nervous system outflow, producing most of the circulating adrenaline. We recently found that the rhesus monkey adrenal gland expresses several genes in a 24-h rhythmic pattern, including TH (the rate-limiting enzyme in catecholamine synthesis) and Atf5 (a transcription factor involved in apoptosis and neural cell differentiation) together with the core-clock genes. To examine whether these core-clock genes play a role in adrenal circadian function, we exposed rat pheochromocytoma PC12 cells to a serum shock and found that it triggered rhythmic oscillation of the clock genes rBmal1, rPer1, rRev-erb{alpha}, and rCry1 and induced the circadian expression of Atf5 but not TH. Furthermore, we found that the CLOCK/brain and muscle Arnt-like protein-1 (BMAL1) heterodimer could regulate Atf5 expression by binding to an E-box motif and repressing activity of its promoter. The physiological relevance of this interaction was evident in Bmal1 –/– mice, in which blunted circadian rhythm of Atf5 mRNA was observed in the liver, together with significantly higher expression levels in both liver and adrenal glands. Although we found no compelling evidence for rhythmic expression of TH in chromaffin cells being regulated by an intrinsic molecular clock mechanism, the Atf5 results raise the possibility that other aspects of chromaffin cell physiology, such as cell survival and cell differentiation, may well be intrinsically regulated.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN MAMMALS, CIRCADIAN rhythms characterize the temporal pattern of secretion of many hormones, including those secreted by the adrenal gland. Recent data demonstrated the circadian expression of clock genes in the adrenal cortex as well as the adrenal medulla, indicating that circadian oscillators are harbored in both regions of this organ (1, 2, 3, 4). The molecular mechanisms underlying the circadian clock involve transcriptional-translational feedback loops that generate molecular oscillations with a period of about 24 h. Within one of the loops, the transcriptional regulators CLOCK and brain and muscle Arnt-like protein-1 (BMAL1) dimerize and bind to E-box motifs in the promoter regions of various genes, including Per1, Per2, Per3, Cry1, and Cry2. In turn, these proteins form period (PYR)/cryptochrome (CRY) heterodimers that inhibit the activity of the CLOCK/BMAL1 complex, thus closing the loop and generating circadian rhythms in their own expression (5, 6). In addition to their role within molecular oscillators, many clock components regulate the expression of effector genes, also called clock-controlled genes (CCGs), through which the clock exerts temporal control of specific cellular processes (7, 8, 9).

Adrenal medulla chromaffin cells are specialized sympathetic cells that synthesize a variety of catecholamines, including adrenaline, noradrenaline, and dopamine, and play a key role in mediating physiological stress responses. In addition, these cells retain a certain degree of pluripotency and have been used as a model to study neuronal differentiation (10, 11). Because the adrenal medulla comprises 20% of the entire gland, pheochromocytoma-derived cell lines have been generated to study chromaffin cell functions. Among the most common cell lines, rat PC12 cells have been largely used as an in vitro model to study both chromaffin cell neuroendocrine physiology and neural plasticity (12, 13).

Recently, we used gene microarrays to profile rhythmic gene expression in the rhesus monkey adrenal gland and found that many genes that are relevant to chromaffin cell physiology had a clear-cut 24-h expression pattern (14). Among those genes, we identified TH [tyrosine hydroxylase (TH)] and Atf5 [activating transcription factor (ATF) 5] as potential clock outputs. A diurnal expression profile has been previously observed for TH, the rate-limiting enzyme in catecholamine synthesis, in the spinal cord and motor regions of the brain (15, 16). Also, an E-box enhancer element in the TH gene promoter has been reported (17, 18), further suggesting that expression of TH could be regulated by elements of the circadian clock. The second candidate, Atf5, is a transcriptional regulator of the ATF/cAMP response element-binding protein (CREB) family, which is involved in apoptosis and neural differentiation (19). Atf5 mRNA has been identified in different circadian microarray studies in both liver and adrenal glands (14, 20, 21, 22, 23), thereby constituting a likely clock output.

The goal of the present study was to elucidate whether a molecular clock within the chromaffin cell is involved in temporal regulation of TH, a gene involved in a tissue-specific function, vs. Atf5, a gene involved in cell survival and differentiation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture conditions
PC12 cells were maintained at 37 C with 5% CO2, in RPMI 1640 (Sigma-Aldrich, St. Louis, MO) medium containing 10% fetal calf serum, 1 mg/ml glucose, 100 U/ml penicillin, and 100 µg /ml streptomycin. Approximately 5 x 104 cells were plated on poly-L-lysine-coated six-well culture plates 5–6 d before the experiments. For the serum shock experiment, 24 h before the treatment, the cells were washed three times with serum-free (SF) medium and then maintained in SF medium. At time zero, the medium was replaced with either medium containing 50% horse serum or SF medium in the case of sham experiments, and after 2 h, this medium was replaced with SF medium. For the dexamethasone experiment, at time zero, dexamethasone (10 mM in ethanol) (Sigma-Aldrich) was added to the medium to a final 100 nM concentration. After 1 h incubation, the cells were washed twice and then maintained in SF medium. At the indicated time points, the cells were washed with ice-cold PBS twice, harvested in lysis buffer (RNeasy kit; QIAGEN, Valencia, CA) plus ß-mercaptoethanol and stored at –80 C.

Preparation of RNA samples and RT-PCR
Total RNA was extracted using the RNeasy kit (QIAGEN) according to the manufacturer’s instructions. For semiquantitative PCR, cDNA samples were synthesized from 2 µg total RNA using the Omniscript RT kit (QIAGEN) and oligo d(T)15 primers (Promega, Madison, WI). The reaction was performed according to the manufacturer’s instructions, in a reaction volume of 20 µl, at 37 C for 1 h. PCR amplifications were performed using 1 µl cDNA, 200 µM dNTPs (Promega), 0.5 µM of each primer, and 2.5 U HotStar Taq polymerase (QIAGEN) in a reaction volume of 25 µl. Reactions were performed according to manufacturer’s instructions (QIAGEN). The number of PCR cycles was established after testing a range of 20–35 cycles to ensure that the amount of DNA product remained in the logarithmic range of the amplification curve. The following primers (Invitrogen, Carlsbad, CA) were used: rat (r) Bmal1 forward, 5'-GCTCCAGCCCACTGAACATCAC-3', and rBmal1 reverse, 5'-CTACAGTGGCCATGGCAAGTCAC-3'; rPer1 forward, 5'-GAACTGGGTGCTGTGCACTCCT G-3', and rPer1 reverse, 5'-AGCTGGACTGGAAGAGC TCCCAC-3'; rCry1 forward, 5'-GCCTGTCCTAAGA GGCTTCCCTG-3', and rCry1 reverse, 5'-ACTGAGGCCAGTGCCCATGGAGC-3'; and rTBP forward, 5'-GTGTTGA CCCACCAGCAGTTCAG-3', and rTBP reverse, 5'-CCTGCAGTAGGACACACAGTGTC-3'. PCR product were separated on 2% agarose gels and visualized by ethidium bromide staining.

Real-time PCR
The RNA was diluted to 0.1 µg/µl, and cDNA was prepared by random-primed RT using random hexamer primers (Promega), 200 ng RNA, and the Omniscript kit (QIAGEN). The RT reaction was diluted 1:100 for PCR analysis. The PCR were prepared as previously described (12). The following primers (Invitrogen) and probes (Sigma Genosys, St. Louis, MO) were used: rTBP (accession no. NM_001004198) forward, 5'-TACAGGTGGCAGCATGAAGTG-3', and reverse, 5'-AAGTAGCAGCACAGAGCAAGCA-3', and probe, 5'-VIC-TCCCTCCTCTGCACTGAGATCACCCT-TAMRA-3'; rRev-erb{alpha} (accession no. NM_145775) forward, 5'-ACCCTGAACAACATGCATTCC-3', and reverse, 5'-GGAGAGAGAAGTG CAGAGTTCGA-3', and probe, 5'-FAM-CTGCCGC TGCCCCCTTGTACA-TAMRA-3'; rTH (accession no. NM_012740) forward, 5'-AGCTTCAATGACGCCAAGGA-3', and reverse, 5'-AGTACGTCAATGGCCAGTGTGT-3', and probe, 5' FAM-TCCAGCGCCCATTCT CTGTGAAGTTT-TAMRA-3'; and rAtf5 (accession no. NM_172336) forward, 5'-GCCCCTCTGCCTTCACTCT-3', and reverse, 5'-GCAGCTGACTTATTCTGGTCTCTCT-3', and probe, 5'-FAM-CTAGTCCTGCCAGCACCCGAGG-TAMRA-3'. The amplification was performed in an ABI/Prism 7700 sequence detector system (Applied Biosystems, Foster City, CA). After PCR was completed, baseline and threshold values were set to optimize the amplification plot. Standard curves were drawn on the basis of the log of the input RNA vs. the critical threshold cycle, which is the cycle in which the fluorescence of the sample was greater than the threshold of baseline fluorescence. Standard curve functions were used to convert the critical threshold values into relative RNA concentrations for each sample. Normalization for both the reference dilution series and experimental samples was performed by running two sets of reactions, one for the target gene and another one for rTBP, the endogenous control. Relative abundance values were calculated for the target mRNA as well as for rTBP as described above. Normalized values were obtained by dividing the relative abundance of the target mRNA by the relative abundance of rTBP.

Analysis of mouse (m)Atf5 gene expression and RNA abundance calculations in liver samples of control and Bmal1–/– mice were performed by real-time RT-PCR using mAtf5-specific MGB probe (Applied Biosystems; Mm00459515_m1) as previously described (20).

Immunocytochemistry
PC-12 cells were seeded in 12-well plates containing glass coverslips coated with Matrigel (BD Biosciences, Franklin Lakes, NJ), and grown until they reached 80% confluency. After a serum shock, the coverslips were collected every 6 h, washed with PBS, and fixed in ice-cold methanol for 10 min. Cells were blocked and incubated overnight with anti-PER1 antiserum (1177, generously provided by Dr. D. R. Weaver) at 4 C. Subsequently, the cells were incubated with a secondary antibody (Invitrogen), and stained for nuclear visualization. Photomicrographs were obtained using a DeltaVision deconvolution microscope (Applied Precision Llc, Issaquah, WA).

Plasmid constructs and genomic cloning
The expression vectors for mClock and mBmal1, together with the E54-TK construct were a gift from Dr. S. Honma (Hokkaido University Graduate School of Medicine, Sapporo, Japan). In silico analysis of 2.0 kb of the predicted 5' flanking region of the rAtf5 gene (GenBank accession no. NW_047558, nucleotides 2826–4826) was performed using the Transcription Element Search System tool (http://www.cbil.upenn.edu/cgi-bin/tess/tess). A 1634-bp DNA segment containing the putative E-box sequence was PCR amplified with specific primers (5'-ATATGGTACCGCAGCACTCTAGCCAATACTC-3' and 5'-ATATAAGCTTGACGAGATCGCGGCTACAG-3') using the FailSafe PCR System (Epicenter Biotechnologies, Madison, WI) and a step-down PCR protocol. The single PCR product obtained was purified (PCR purification kit; QIAGEN), cloned into a pGEMT vector (pGEM*-T Easy; Promega), and then excised by HincII-HindIII digestion, subcloned into the SmaI-HindIII sites of the reporter vector pGL2-Basic-luciferase (Promega), and verified by sequencing.

Site-directed mutagenesis
We generated a mutant construct by deleting the two central nucleotides of the rAtf5 E-box element (CACGTG->CA**TG) in the rAtf5 putative promoter by site-directed mutagenesis (QuikChange site-directed mutagenesis kit; Stratagene, La Jolla, CA). The 50-µl reactions were performed according to the manufacturer’s instructions using the following primers: forward, 5'-CAGACCTG TCACCACATGGTGATGGTGAAGGCC-3', and reverse, 5' GGCCTTCACCATCACCATGTGGTGACAGGTCTG-3'. The PCR program used consisted of an initial activation step of 2 min at 95 C, followed by 18 cycles of denaturing at 94 C for 2 min, annealing at 60 C for 2 min, extension at 68 C for 7.5 min, and a final extension of 7 min at 68 C. The mutation was verified by sequencing.

EMSA
PC12 cells were seeded at a density of 60–70% in 10-cm diameter dishes. On the following day, the cells were cotransfected with 10 µg mClock and mBmal1 using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instruction. The cells were harvested 24 h after transfection, nuclear extracts were prepared as previously described (24), aliquoted, and stored at –80 C.

The oligonucleotides (Invitrogen) used were as follows: mPer1 E-box sense, 5'-CA CCCAAGTCCACGTGCAGGGATGTG-3', and antisense, 5'-CACATCCCTGCACGTGGACT TG GGTG-3'; rTH E-box sense, 5'-ATTCAGAGGCAGGTGCCTGTGACAG TG-3', and antisense, CACTGTCACAGGCACCTGCCTCTGAAT-3'; rAtf5 E-box sense, 5'-CCTGTCACCACACGTGGTG ATGGTGA-3', and antisense, 5'-TCACCATCACC ACGTGTGGTGACAGG-3'; and rAtf5 mutated E-box sense, 5'-CCTGTCACCACAATTGGTGATGGTGA-3', and antisense 5'-TCACCATCACCAATTG TGGTGACAGG-3' (E-box elements are in bold, and mutated nucleotides are underlined). Double-stranded probes were generated by labeling 50 pmol DNA with 100 µCi [{gamma}-32P]ATP (MP Biomedicals, Irvine, CA), using T4 polynucleotide kinase (Promega) according to the manufacturer’s instructions. Binding reactions were conducted for 30 min at room temperature using 8 µg nuclear protein and 20,000 cpm radiolabeled probe. Where indicated, unlabeled DNA was added to the binding reaction as competitor. An anti-BMAL1 antibody (Alpha Diagnostics International, Inc., San Antonio, TX) was added to the indicated samples before the addition of the radiolabeled probes. The complexes were resolved by electrophoresis on a 5% nondenaturing polyacrylamide gel (0.25x Tris-borate-EDTA buffer) at 100 V for 2–3 h. Gels were dried and exposed to film at –80 C overnight.

Promoter assays
PC12 cells were seeded at a density of 500,000 cells per well in six-well plates. Twenty-four hours later, transfections were performed using Lipofectamine 2000 (Invitrogen). To verify the transcriptional activity of the rAtf5 putative promoter, different concentrations of pGL2-ATF5p (250–1000 ng/well) were transiently transfected together with 20 ng/ml of a reporter plasmid constitutively expressing ß-galactosidase (CMV-Sport ß-gal; Invitrogen), which was used to normalize for transfection efficiency. To examine the ability of CLOCK/BMAL1 to transregulate the rAtf5 promoter, PC12 cells were cotransfected with 500 ng wild-type (WT) pGL2-ATF5p or mutant pGL2-ATF5p (pGL2-ATF5p'), together with increasing amounts of mClock- and mBmal1-expressing constructs (125–500 ng each). Transactivation of the mPer1 promoter was assessed using 200 ng of the E54-TK construct in combination with 125 ng mClock and mBmal1 constructs. In all cases, control experiments were performed by transfecting the empty reporter vectors pGL2 or pGL3. As before, cells were collected 24 h later. In all the experiments, the cells were collected 24 h after transfection and assayed for luciferase and ß-galactosidase activity, as reported previously (25).

Animals
Eighteen WT and 18 Bmal1–/– mice on C57BL/6J background (10 backcross generations) were produced through heterozygous matings and genotyped by PCR. Animals were synchronized to a 12-h light, 12-h dard cycle for at least 2 wk before transfer to constant darkness (DD). Liver tissue samples were collected at 4-h intervals beginning after 34 h of exposure to DD, immediately frozen on dry ice, and stored at –80 C until RNA extraction; three animals of each genotype were used per time point. All animal studies were conducted in accordance with the regulations of the Committee on Animal Care and Use at the Cleveland Clinic Foundation.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Rat pheochromocytoma PC12 cells contain a functional molecular clock
To verify that the rat pheochromocytoma PC12 cell line is an appropriate model in which to investigate circadian mechanisms of adrenal chromaffin cells, we first used RT-PCR to confirm that the core mammalian clock genes, including rClock, rBmal1, rPer1, rPer2, rCry1, and rRev-erb{alpha} are expressed in these cells (data not shown). To determine whether the mRNAs encoded by clock genes accumulate with circadian rhythmicity in these cells, as reported previously in other cell lines (26, 27, 28), we measured their expression after a 50% horse serum shock using real-time PCR and semiquantitative RT-PCR. The mRNA levels of rRev-erb{alpha}, rBmal1, rPer1, and rCry1 oscillated with approximately 20- to 24-h periodicity after the serum shock, whereas expression of rTBP, a housekeeping gene, remained constant (Fig. 1AGo). The oscillations occurred with phase relationships similar to those of a typical circadian clock, so that the oscillation of rBmal1 was in antiphase with that of rPer1 and rRev-erb{alpha}, whereas rCry1 peaked approximately 4–8 h before rPer1. In agreement with previous results obtained in rat-1 fibroblasts (26, 29), rBmal1 levels acutely increased 4 h after the serum shock, whereas the levels of rPer1 remained low initially and then increased gradually during an initial 20-h period (Fig. 1AGo). Importantly, in control experiments, in which the cells were transferred to SF medium, no oscillations in rRev-erb{alpha} (Fig. 1AGo) or rBmal1 mRNA (data not shown) were detected in this cell line. To further characterize the effect of a serum shock on the molecular clock mechanism of PC12 cells, we used immunocytochemistry to analyze changes in the subcellular localization of rPER1. Although the cytoplasmic staining intensity remained similar at different times of the day, the nuclear staining showed an oscillatory pattern that was characterized by an increase in nuclear signal between 12 and 18 h after the serum shock, reaching 48% of the nuclei at 24 h, i.e. approximately 4 h after the peak of rPer1 mRNA (Fig. 1BGo).


Figure 1
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  		FIG. 1. Circadian expression of clock genes in rat pheochromocytoma PC12 cells. A, A 50% horse serum shock (SS) but not SF medium triggers the circadian expression of rRev-erb{alpha} (Taqman PCR). Circadian expression of rBmal1, rPer1, and rCry1 after the SS was measured using RT-PCR. Expression levels were normalized to those of rTBP, an endogenous control. Results are the mean ± SEM of four independent samples, expressed in relative units. The data have been normalized such that the average signal intensity across all time points is 1. A representative semiquantitative RT-PCR result is shown for each mRNA. B, PER1 subcellular localization after a serum shock. Representative immunocytochemistry. White arrow, Cytoplasmic staining; white arrowhead, nuclear staining. The percentage of PER1-positive nuclei is expressed as the mean of three experiments.

 
A serum shock induces circadian expression of rATF5 but not of rTH in PC12 cells
In a previous study, we identified TH and Atf5 as genes showing a diurnal rhythm of expression in the adrenal gland, suggesting that they may be potential targets of an intrinsic molecular clock (12). In support of this hypothesis, the rat TH promoter has an E-box consensus motif CAGGTG adjacent to an activator protein 1 (AP-1) binding site at position –191 bp from the transcription starting site (11), which represents a potential Clock/Bmal1 binding site (Fig 2AGo). To identify potential Clock/Bmal1 binding sites in the 5'-flanking region of the rat Atf5 gene, we analyzed a 2.0-kb genomic region upstream of the Atf5 transcription start site reported by Hansen et al. (30). Using the Transcription Element Search System software, we found two potential CAAT-boxes, at positions –495 and –585 bp, respectively, and a TATA-like sequence at –404 bp. These results provided support for the idea that this region plays a role in the regulation of Atf5 expression. Importantly, in addition to potential binding sites for several transcriptional regulators (AP-1, CREB, glucocorticoid receptor, footprint II binding factor, nuclear factor-erythroid derived 2, GATA-binding protein, and specificity protein 1), we identified a putative Clock/Bmal1 consensus E-box CACGTG at position –1385 (Fig. 2AGo).


Figure 2
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  		FIG. 2. A serum shock triggers the circadian expression of rAtf5 in PC12 cells. A, Schematic representation of a 450-bp region of the rat TH promoter displaying the E-box element and the 5'-flanking region of the rat Atf5 gene. A potential TATA box at position –404, two potential CAAT boxes, and putative SP1, GR, CRE, GATA-1, NF-E2, and F2F motifs are indicated. An E-box element is present at –1385. In both graphics, position +1 indicates the transcriptional start sites. B, A serum shock (SS) does not trigger rTH mRNA rhythmic expression. In a control experiment, the gene is activated by 1 h exposure to 100 nM dexamethasone (Dex). C, A serum shock (SS) induces the circadian expression of rAtf5. Expression levels were measured by Taqman PCR and normalized to the levels of rTBP. Results are the mean ± SEM of four (SS) and three (Dex) independent samples, expressed in relative units.

 
Because both rTH and rAtf5 are expressed in PC12 cells (18, 31), we hypothesized that if these genes are regulated by Clock and Bmal1, then synchronization of the cellular clocks should also induce circadian oscillations in rTH and rAtf5 mRNA expression. We measured rTH and rAtf5 mRNA levels in the same serum shock experiments illustrated in Fig. 1AGo, in which synchronization of clock genes was observed. The serum shock failed to trigger circadian oscillations in rTH mRNA levels. Instead, an acute but insignificant increase was observed at 4 h, followed by a steady increase through 40 h (Fig. 2BGo). To verify that the promoter of the rTH gene is responsive to a known activating stimulus, we incubated the cells with dexamethasone, a glucocorticoid receptor agonist (32), and measured the levels of rTH mRNA during a 44-h period. After a 1-h exposure to dexamethasone (100 nM), rTH mRNA levels increased, reaching a maximum at 4 h, and then decreased to basal levels after 36 h. In contrast to rTH, the serum shock induced robust circadian expression of rAtf5, with a periodicity of 20–24 h (Fig. 2CGo), suggesting that this gene may be controlled by the intrinsic molecular clock.

CLOCK and BMAL1 bind to the Atf5 E-box but not to the TH E-box
To determine whether CLOCK and BMAL1 are able to bind the rTH and rAtf5 E-boxes, we cotransfected constructs expressing mCLOCK and mBMAL1 into PC12 cells and performed EMSAs using double-stranded oligonucleotide probes containing the E-boxes of mPer1, rTH, rAtf5, or a mutated version of the rAtf5 E-box. As expected, the CLOCK/BMAL1 heterodimer strongly bound to the mPer1 E-box probe (Fig. 3AGo). The EMSAs also show that basal binding activity was present when nuclear extracts from nontransfected (naive) PC12 cells were used, probably due to endogenous CLOCK/BMAL1 dimers. The specificity of the signal was demonstrated by the ability of an antibody that immunoreacts with the DNA-binding domain of BMAL1 to blunt the binding of CLOCK/BMAL1 to the mPer1 E-box (Fig. 3AGo). When nuclear extracts from naive PC12 cells were incubated with a probe containing the rTH E-box within the context of a dyad element and only a portion of the adjacent AP-1 site, a weak signal was observed, the intensity of which did not change when the Clock and Bmal1 constructs were cotransfected (Fig. 3BGo, left panel). When used in competition experiments, a molar excess of the unlabeled rTH E-box probe did not reduce the binding of CLOCK/BMAL1 to the mPer1 E-box probe (Fig. 3BGo, right panel), indicating that the rTH E-box is not recognized by this transcriptional complex.


Figure 3
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  		FIG. 3. CLOCK and BMAL1 bind to the mPer1 E-box but not the rTH E-box in PC12 cells. Nuclear protein extracts from either naive PC12 cells or PC12 cells cotransfected with mClock and mBmal1 were incubated with the specified probes. A, Binding of CLOCK/BMAL1 to the mPer1 E-box. An antibody against the DNA-binding domain of BMAL1 was used to assess the presence of BMAL1 in the complexes. A representative EMSA is shown. B, Left panel, the signal obtained with the rTH E-box does not change upon CLOCK/BMAL1 expression (representative EMSA shown); right panel, the rTH E-box does not compete with the mPer1 E-box for the CLOCK/BMAL1 complex. The rTH E-box was used as a cold competitor at a 200-fold molar excess. The major specific complexes are indicated by a black arrow.

 
When the rAtf5 E-box probe was incubated with naive PC12 cells extracts, a complex similar in size to that obtained when using the mPer1 E-box was observed mainly in nuclear fractions. The intensity of that basal signal increased robustly when Clock and Bmal1 constructs were cotransfected and was blocked by the anti-BMAL1 antibody (Fig. 4AGo), indicating that the CLOCK/BMAL1 dimer recognizes the rAtf5 E-box. The signal was abolished by a fold molar excess of unlabeled rAtf5 E-box (Fig. 4AGo) but not by a fold molar excess of an unlabeled mutated rAtf5 E-box oligonucleotide (CAATTG), further confirming the specificity of the binding (Fig. 4BGo). Moreover, in competition experiments using the mPer1 E-box probe, a fold molar excess of unlabeled rAtf5 E-box oligonucleotide strongly competed with the binding of CLOCK/BMAL1, whereas a fold molar excess of the unlabeled mutant had no effect (Fig. 4BGo). In a positive control experiment, a fold molar excess of unlabeled mPer1 E-box abolished the signal of the mPer1 E-box probe (Fig. 4BGo).


Figure 4
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  		FIG. 4. CLOCK and BMAL1 bind to the Atf5 E-box. Nuclear protein extracts from either naive PC12 cells or PC12 cells cotransfected with mClock and mBmal1 were incubated with the specified probes. A, Binding of CLOCK/BMAL1 to the rAtf5 E-box element. A representative EMSA is shown. Specificity was tested using a mutant rAtf5 E-box [N (n)CACGTG N(n) -> N (n)CAATTG N (n)] as a cold competitor. B, The rAtf5 E-box element competes with the mPer1 E-box for the CLOCK/BMAL1 heterodimer. Representative competition assays used the radiolabeled mPer1 E-box and the mPer1 E-box and rAtf5 E-box and mutant rAtf5 E-box as cold competitors. The black triangles represent 20- and 200-fold molar excess of the indicated cold oligonucleotide.

 
The CLOCK/BMAL1 heterodimer regulates the rAtf5 promoter via the E-box site
To investigate whether the CLOCK/BMAL1 heterodimer regulates the transcriptional activity of the rAtf5 promoter, we cloned a 1634-bp fragment from the 5' region of the rAtf5 gene into a luciferase reporter plasmid. A dose-dependent increase in luciferase activity was observed in transient transfection experiments in PC12 cells, indicating that the fragment cloned is transcriptionally active (Fig. 5AGo). When this construct was cotransfected together with Clock and Bmal1, CLOCK/BMAL1 consistently repressed the luciferase activity in both PC12 and NIH3T3 cells in a dose-dependent manner (respectively, Fig. 5BGo and supplemental Fig. 1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). To examine whether this negative transcriptional effect was exerted through the E-box element, we coexpressed CLOCK and BMAL1 in PC12 cells together with the above described mutated version of the rAtf5 promoter. When the mutated rAtf5 promoter construct was used, the repressive effect of CLOCK/BMAL1 was abolished, indicating that the heterodimer acts via the E-box site (Fig. 5CGo). As a control for normal functioning of the ectopically expressed mCLOCK and mBMAL1, we cotransfected them with the E54-TK construct (33), which contains the 5' flanking region of the mPer1 gene including three E-box elements, and verified a transactivation of this promoter (Fig. 5DGo), as previously reported (33).


Figure 5
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  		FIG. 5. rAtf5 promoter is negatively regulated by CLOCK and BMAL1. A, The 1.63-kb from the rat Atf5 gene 5'-flanking region activates gene expression. A dose-response experiment was performed transfecting the luciferase reporter construct containing the E-box element (pGL2-ATF5p) into PC12 cells at the indicated doses. B, CLOCK and BMAL1 down-regulate rat Atf5 promoter activity in cotransfection experiments in PC12 cells. C, A deletion within the E-box [N (n)CACGTG N(n) -> N (n)CA... TG N (n)] blocks the effect of CLOCK and BMAL1 on the transcriptional activity. D, A control experiment was performed using 200 ng of the E54-TK reporter construct, containing three E-boxes of the mPer1 promoter in tandem cotransfected with mClock and mBmal1 in PC12 cells. In all the experiments, the cells were harvested 24 h after transfection for luciferase and ß-galactosidase assays. Results are the mean ± SEM of six (A, B, and D) or four (C) samples per group.

 
Atf5 is a target of the circadian clock in vivo
The in vitro results from the present study strongly suggest that Atf5 gene expression is directly repressed by CLOCK/BMAL1. If this mechanism operates in vivo, then in animals with targeted disruption of the Bmal1 gene, in which the circadian clock is impaired, not only should circadian expression of Atf5 be abolished but also the mRNA levels should be elevated. We tested this hypothesis using both the liver and the adrenal gland of Bmal1–/– mice. The liver was chosen because robust circadian expression of Atf5 has been previously reported to occur in this organ (18, 19, 20) and binding to the Atf5 E-box follows a circadian pattern in WT animals (supplemental Fig. 2, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). We measured Atf5 mRNA levels across 24 h in WT and Bmal1–/– mice maintained under DD. As expected, the circadian rhythm in Atf5 expression observed in WT mice was blunted in Bmal1–/– mice (Fig. 6AGo). Importantly, the levels of Atf5 mRNA remained higher in Bmal1–/– compared with WT animals across 24 h, the overall daily expression being approximately 2.2-fold higher (P < 0.05) (Fig. 6BGo). Likewise, expression of Atf5 was significantly higher in the adrenal glands of Bmal1–/– compared with WT animals at zeitgeber time 06, the time point corresponding to the maximum observed in the liver (P < 0.05) (Fig. 6CGo). Altogether, these in vivo data support the hypothesis that CLOCK/BMAL1 negatively regulates Atf5 gene expression.


Figure 6
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  		FIG. 6. Atf5 is up-regulated in the liver of Bmal1–/– mice. A, Circadian expression of mAtf5 is abolished in Bmal1–/– mice. Expression was measured using quantitative Taqman PCR. Results are expressed as the mean ± SEM of three animals per time point. *, P < 0.05 vs. WT at 50 h. B, Average daily levels of mAtf5 mRNA are 2.3-fold increased in Bmal1–/– mice compared with WT. Levels of mAtf5 mRNA were calculated as the mean ± SEM of all time points across the day. *, P < 0.05. C, Levels of mAtf5 mRNA are 2.7-fold higher in Bmal1–/– mice adrenal glands at zeitgeber time 06 (38 h in DD). Results are expressed as the mean ± SEM of six animals per group. *, P < 0.05. In A–C, levels of mAtf5 mRNA were normalized against the levels of ß-macroglobulin.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we used PC12 cells to study the role of the molecular clock in chromaffin cells. We found that a serum shock triggers the circadian expression of rRev-erb{alpha}, rBmal1, rCry1, and rPer1 in those cells, with phase relationships expected for the circadian oscillator. Also, the treatment induced synchronized translocation of rPER1 into the nucleus, an event that is necessary for the regulation of CLOCK/BMAL1 transcriptional activity. Altogether, those data confirmed that a functional clock is present in this cell line, making it suitable for the study of CCGs.

Our study focused on two potential clock outputs that represent different aspects of the chromaffin cell biology. The first encodes TH, an enzyme that catalyzes the major rate-limiting step in the synthesis of catecholamines (34). Previous data showed the existence of diurnal rhythms in both TH activity and TH expression in several tissues (15, 16, 35, 36). These data, together with the finding that an E-box is present in the TH promoter (17, 18), raised the possibility that TH may be directly regulated by the CLOCK/BMAL1 heterodimer. Our in vitro results do not support this hypothesis, however. Although we found that the expression of circadian clock components could be synchronized by a serum shock, TH mRNA levels did not oscillate, as would be expected for a CCG. In addition, the EMSA results indicate that the CLOCK/BMAL1 heterodimer does not bind to the TH E-box. Altogether, these data suggest that TH is not a CCG.

The diurnal pattern of TH gene expression observed in vivo could alternatively stem from circadian cues originating in the SCN and being conveyed to the chromaffin cells via the splanchnic nerve. Stimulation of this nerve likely triggers calcium influx into chromaffin cells, causing the subsequent activation of CRE and AP-1 sites located on the TH promoter (37, 38). Thus, rhythmic neural activation could translate into rhythmic expression of the TH gene. An alternative plausible mechanism could involve the circadian adrenocortical secretion of glucocorticoids, which are known to have major effects on the medulla. Oscillating levels of glucocorticoids could activate the glucocorticoid receptor element located in the TH promoter (39) in a rhythmic fashion and thus regulate temporal expression of the TH gene.

ATF5 (also known as ATFx), is a bZIP transcription factor from the CREB/ATF family of transcriptional regulators that is involved in apoptosis as well as differentiation of neural progenitor cells and chondroblasts (40, 41, 42). Circadian expression of Atf5 has been found to occur in mouse liver (20, 21, 22) as well as in serum-shocked rat 3Y1 fibroblasts (43). In the present study, Atf5 mRNA displayed a robust circadian pattern of accumulation in PC12 cells after the serum shock, which persisted for at least 44 h. Analysis of the rat Atf5 promoter revealed the existence of a canonical clock E-box at position –1385 bp from the transcription start site, which was recognized by CLOCK/BMAL1 heterodimers in gel shift assays. Functional analysis of Atf5 5'-flanking region showed that the promoter activity was repressed by CLOCK/BMAL1 dimers through the E-box element. In support of our in vitro results, the in vivo data demonstrated that in the absence of BMAL1, the circadian oscillation of Atf5 mRNA is abolished in the liver, whereas its levels are significantly higher at all time points. Likewise, the results showed that Atf5 gene expression is higher in the adrenal glands of Bmal1–/– mice, although in this case only one time point was assessed, due to a limited availability of Bmal1–/– mice. Altogether, these data strongly suggest that Atf5 is a direct target of the circadian clock.

The use of genome-wide analysis of circadian gene expression in different species has revealed that although a significant percentage of the mammalian transcriptome is under circadian control (20, 21, 22), only a small percentage of the genes are first-order CCGs, i.e. directly regulated by the molecular clock (20). Hence, most of the genes expressed with a circadian rhythm are second-order CCGs, indirectly regulated by the molecular clock through circadian transcriptional cascades that involve first-order CCGs. In this context, regulation of Atf5 could confer the clock control over a number of second-order CCGs that are in turn regulated by this transcription factor. Furthermore, because ATF5 homodimers bind to the CRE motif (44), a regulatory element that is widely present in gene promoters, the circadian signal could be transmitted to a large number of downstream genes.

Transcriptional repression by CLOCK/BMAL1 has been previously reported for c-myc and TTF-1, presumably through E-box elements located in their promoters (45, 46, 47). A recent study demonstrated that a dual role for this circadian complex is feasible upon binding of CRY1, a clock component that can switch the heterodimer function from transcriptional activator to transcriptional repressor (48). The negative action of CLOCK/BMAL1 on Atf5 supports a hypothesis proposed in the mentioned study, that the role of this heterodimer may vary depending on the basal activity of the promoter, so that for very active promoters, additional activation by CLOCK/BMAL1 would not induce a significant change, whereas repression could result in greater regulation (48). It is also possible that, as in the case of the Cry1 gene, repression of Atf5 promoter activity involves the recruitment of CRY1, which, by the time the promoter activity was measured, could have been synthesized and forming complexes with CLOCK/BMAL1.

The adrenal medulla is a dynamic tissue with a large regenerating and differentiating capacity that can undergo morphological as well as biochemical changes in response to environmental stress (49, 50). Adrenal chromaffin cells can quickly regenerate after medulla injuries, by differentiation of preexisting pheochromoblasts (51, 52). In PC12 cells, down-regulation of Atf5 is required for nerve growth factor (NGF)-induced differentiation into sympathetic neurons (31, 41), whereas in vivo, the loss of Atf5 expression appears to be necessary for differentiation of neurons, astrocytes, and oligodendrocytes (41, 44, 53). These findings suggest that expression of Atf5 is required for maintenance of neural progenitor cell cycling, whereas its down-regulation leads to differentiation. In this context, the repressing effect of CLOCK/BMAL1 on Atf5 expression suggests that perhaps the heterodimer plays a role in the down-regulation of Atf5 during differentiation. A role for clock genes in neuronal differentiation is certainly plausible, given that clock components are involved in the differentiation of other tissues (8, 9, 54). One possibility is that neurotrophic factors released in an autocrine manner by chromaffin cells or in a paracrine manner by autonomic preganglionic neurons innervating the adrenal medulla may regulate this process. In support of this hypothesis, intracerebroventricular administration of NGF has been shown to induce circadian phase shifts in hamsters together with activation of ERK1/2 and induction of c-Fos expression in the suprachiasmatic nuclei, demonstrating that this factor has a direct effect on the circadian system (55). Thus, it is tempting to speculate that NGF could have an effect on the molecular clock, which in turn could affect Atf5 expression.

In summary, the present data confirm the existence of a molecular clock in a model of chromaffin cell and demonstrate that the transcription factor Atf5 is a CCG, being directly down-regulated by the CLOCK/BMAL1 heterodimer. Although a function for the chromaffin cell clock in the regulation of catecholamine synthesis was not disclosed, a role in regulation of adrenal medulla homeostasis, particularly in processes such as differentiation and control of apoptosis is proposed.


   Acknowledgments
 
We thank Dr. Alejandro Lomniczi and Dr. Cecilia Garcia Rudaz for technical advice and Dr. Bredford Kerr and Dr. Roman Kondratov for their critical comments on the manuscript.


   Footnotes
 
This work was supported by National Institutes of Health Grants AG-029612, HD-29186, and RR-00163 (to H.F.U.), GM075226 (to M.P.A.), and HD 25123 (to S.R.O.).

Disclosure Summary: D.R.L., L.G., L.T., M.P.A., S.R.O., and H.F.U. have nothing to declare.

First Published Online September 6, 2007

Abbreviations: AP-1, Activator protein 1; ATF, activating transcription factor; BMAL1, brain and muscle Arnt-like protein-1; CCG, clock-controlled gene; CREB, cAMP response element-binding protein; CRY, cryptochrome; DD, constant darkness; m, mouse; NGF, nerve growth factor; PER, period; r, rat; SF, serum-free; TH, tyrosine hydroxylase; WT, wild type.

Received May 9, 2007.

Accepted for publication August 27, 2007.


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