Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M413279200 on February 2, 2005

J. Biol. Chem., Vol. 280, Issue 14, 14331-14340, April 8, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
280/14/14331    most recent
M413279200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chiang, M.-C.
Right arrow Articles by Chern, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chiang, M.-C.
Right arrow Articles by Chern, Y.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

cAMP-response Element-binding Protein Contributes to Suppression of the A2A Adenosine Receptor Promoter by Mutant Huntingtin with Expanded Polyglutamine Residues*{boxs}

Ming-Chang Chiang{ddagger}§, Yi-Chao Lee{ddagger}, Chuen-Lin Huang{ddagger}, and Yijuang Chern{ddagger}§

From the {ddagger}Division of Neuroscience, Institute of Biomedical Sciences, Academia Sinica and §Institute of Neuroscience, National Yang Ming University, Taipei 11529, Taiwan

Received for publication, November 24, 2004 , and in revised form, January 28, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Huntington's disease is a neurodegenerative disease resulting from a CAG (glutamine) trinucleotide expansion in exon 1 of the Huntingtin (Htt) gene. The role of the striatum-enriched A2A adenosine receptor (A2A-R) in Huntington's disease has attracted much attention lately. In the present study, we found that expression of mutant Htt with expanded poly(Q) significantly reduced the transcript levels of the endogenous A2A-R in PC12 cells and primary striatal neurons. Cotransfection of various promoter constructs of the A2A-R gene and an expression construct of poly(Q)-expanded Htt revealed that the Htt mutant suppressed the core promoter activity of the A2A-R gene. Stimulation of the A2A-R using CGS21680 forskolin, and a constitutively active cAMP-response element-binding protein (CREB) mutant elevated the reduced promoter activity of the A2A-R gene by mutant Htt. Moreover, the effect of CGS was blocked by an A2A-R-selective antagonist (CSC), two inhibitors of protein kinase A, and two dominant negative mutants of (CREB). The protein kinase A/CREB pathway therefore is involved in regulating A2A-R promoter activity. Consistently, an atypical CRE site (TCCAGG) is located in the core promoter region of the A2A-R gene. Electrophoretic gel mobility shift assay and mutational inactivation further demonstrated the functional binding of CREB to the core promoter region and showed that expression of poly(Q)-expanded Htt abolished the binding of CREB to this site. Stimulation of the A2A-R restored the reduced CREB binding caused by the mutant and concurrently reduced mutant Htt aggregation. Collectively, the poly(Q)-expanded mutant Htt suppressed expression of the A2A-R by inhibiting its core promoter at least partially by preventing CREB binding.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Huntington's disease (HD)1 is an autosomal dominant neurodegenerative disease characterized by chorea, dementia, and psychiatric symptoms. The causative mutation is a CAG trinucleotide expansion in exon 1 of the Huntingtin (Htt) gene. Normal chromosomes have 35 or fewer CAG repeats in the N-terminal region, whereas HD is associated with 36 or more repeats (1). The CAG repeats are translated into polyglutamine residues (poly(Q)) in the Htt protein. When the number of CAG repeats exceeds 36, specific degeneration of several brain areas (e.g. the striatum and cortex) occurs. The reason for such regional susceptibility to mutant Htt with poly(Q) expansion remains unclear. Accumulating evidence from different laboratories suggests that mutant Htt forms aggregates and causes aberrant protein-protein interactions with a number of important transcription factors or coactivators, such as p53, CREB binding protein (CBP), SP1, and TAFII130 (2–6). Alteration of the transcription profiles of cells expressing mutant Htt aggregates is therefore expected (7–12). Sequestration of and/or interference with important transcriptional regulatory proteins have been proposed to be critical mechanisms in HD progression. For example, mutant Htt is known to be associated with the Sp1-TAFII130 complex and causes improper location of RNA polymerase II on the promoter region of the dopamine D2 receptor gene, which subsequently hinders transcription of the gene (5). Moreover, mutant Htt has been shown to act as a transcriptional coactivator of the nuclear hormone receptor (13). Note that the effect of mutant Htt on the overall gene expression profiles appeared to be specific because no changes in the expression of cytoskeletal proteins (including MAP2, glial fibrillary acidic protein, myelin basic proteins, and neurofilaments), enzymes of intermediary metabolism (such as glyceraldehyde phosphate dehydrogenase and glucose-6-phosphate isomerase), caspases, mitochondrial proteins, or proteolytic enzymes were observed in a transgenic HD mouse model (14).

The A2A adenosine receptor (A2A-R) is enriched in Htt/poly(Q)n-sensitive, enkephalin-containing striatal neurons (15–17). We previously cloned cDNA and the gene of the A2A-R, which contains seven transmembrane domains and belongs to the G protein-coupled receptor family (18–22). The A2A-R gene contains two independent TATA-less promoters (P1 and P2), which produce transcripts whose 5'-untranslated regions are of different lengths but that encode the same protein (18, 19). Our previous study demonstrated that P2 is the major promoter of the A2A-R gene, which is active in various tissues including the brain (19, 21). One of the physiological functions of A2A-Rs is to protect cells against various assaults. By using PC12 as a neuron-like model system, we demonstrated that atypical protein kinase Cs function downstream of protein kinase A (PKA) to mediate the protective effect of the A2A-R against apoptosis evoked by serum withdrawal in PC12 cells (23). In addition, A2A-R stimulation rescued the blockage of nerve growth factor-induced neurite outgrowth when the nerve growth factor-evoked mitogen-activated protein cascade was suppressed (6). Moreover, stimulation of the A2A-R was found to delay apoptosis in human neutrophils (24) and to protect the hippocampus from excitotoxicity and the liver and kidney from ischemic injury (25, 26). The role of the A2A-R in HD therefore has also attracted much attention in the past few years (27–30). Specifically, activation of the A2A-R appears to exhibit a dual effect on striatal neurodegeneration caused by a mitochondrial toxin (3-nitropropionic acid) (29). Aberrant amplification of A2A-R signaling has also been demonstrated in striatal cells and in peripheral blood cells expressing mutant Htt (31, 32). Because expression of the striatal A2A-R is markedly reduced during the progression of HD (15, 17, 33), there is still controversy as to whether the A2A-R can serve as a therapeutic target for HD. In the present study, we investigated regulation of the A2A-R gene in the presence of mutant Htt with poly(Q) expansion; we found that mutant Htt suppressed the P2 core promoter activity of the A2A-R gene. In addition, activation of the A2A-R reversed the suppressive effect of mutant Htt through a PKA/CREB-mediated pathway.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Reagents—All reagents were purchased from Sigma except where specified. Forskolin, 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxy-amidoadenosine (CGS21680 CGS), and 8-(3-chlorostyryl) caffeine (CSC) were purchased from Research Biochemical (Natick, MA). DMEM, fetal bovine serum, and horse serum were purchased from Invitrogen. H-89 and KT5720 were from Biomol (Plymouth Meeting, PA).

Constructs—The human Htt exon1 fragment of a normal allele containing the 25 CAG repeat ((Q)25) and that of a mutant Htt allele harboring the 158 CAG repeat ((Q)158) were amplified from genomic DNAs collected from HEK293 cells and a transgenic HD mouse model (R6/2) (34), respectively, by PCR using the following primers: 5'-GGACTGCCGTGCCGGGCGGGAGACCG-3' and 5'-TCGGTGCAGCGGCTCCTCAGCCA-CAG-3'. Fragments were subcloned into the pcDNA3.1/V5-His vector using a TOPO-TA expression kit (Invitrogen) following the manufacturer's protocol. The resultant constructs were designated pcDNA3.1-Htt-(Q)25 and pcDNA3.1-Htt-(Q)158, respectively. The hrGFP cDNA fragment of Vitality® humanized GFP (hrGFP) was removed from the Vitality® hrGFP vector by digestion with NheI/SacI and was subcloned into the NotI site of pcDNA3.1-Htt-(Q)n constructs to produce constructs encoding an N-terminal fragment of Htt with the indicated number of poly(Q) residues fused to hrGFP (designated Htt-(Q)n-hrGFP). Because of the instability of the CAG repeat in bacteria, three pcDNA3.1-Htt-(Q)n-hrGFP constructs containing different numbers of CAG repeats (48, 73, and 109) were created while subcloning the original pcDNA3.1-Htt-(Q)158-hrGFP construct. The pGL2-luciferase reporter constructs, which contain various lengths of the promoter fragment of the rat A2A-R gene, and the CREB-VP16 construct were created as described elsewhere (6, 18). The pRL-SV40 and pRL-TK constructs were purchased from Promega (Madison, WI). The pCMV-{beta}-galactosidase was from Clontech. The expression constructs, which encode a dominant negative S133A-CREB mutant (CREBm1), and a dominant negative CREBR287L mutant, as well as the A2A-R promoter constructs are described elsewhere (18, 35). Promoter constructs harboring mutations at the CRE site (-246 to -241; TCCAGG) were created by PCR using pGL2-A2AR-250/-145 as the template together with the indicated sense primer (M2, 5'-AACATAACCCTCTTGCCCGGTATC-3'; M4, 5'-AACATAACCCTCTTGACCTGTATC-3'; M6, 5'-AACATAACCCTCTTGAACTTTATC-3') and the same antisense primer (5'-CTTTATGTTTTTGGCGTCTTCCA-3'). The resultant PCR products were then subcloned into pGL2-Basic (Promega) following the manufacturer's protocol. Mutations of these promoter constructs were confirmed by DNA sequencing. All plasmids used in the transient transfection experiments were prepared by CsCl gradient purification.

Cell Culture and Transfection—PC12 cells were originally obtained from the American Type Culture Collection (Manassas, VA) and were maintained in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 5% fetal bovine serum (FBS, Invitrogen) plus 10% horse serum (Invitrogen) in an incubation chamber gassed with 10% CO2, 90% air at 37 °C. A123 is a PKA-deficient variant of PC12 cells (36), kindly provided by Dr. J. A. Wagner (Cornell University Medical College, New York, NY). A123 cells were maintained in DMEM supplemented with 5% (v/v) horse serum and 10% (v/v) FBS. Cells were grown on tissue culture plates coated with poly-L-lysine (Sigma). The day before transfection, cells were seeded onto a 35-mm dish at a density of 2 x 105 cells per well. For transfection, cells were grown in wells for 24 h and then exposed to a mixture of 3 µl of Lipofectamine 2000 (LF2000, Invitrogen) and 2 µg of the desired DNAs for 5 h in serum-free DMEM. Next, cells were washed with PBS, followed by one more wash with normal growth medium, and were cultured as described above for another 48 h before the indicated drug treatment(s) for an additional 24 h.

Primary striatal neuron cultures were prepared from brains of E19 SD rats. Briefly, tissues were minced and treated with trypsin (0.1%, Invitrogen) for 10 min at 37 °C. Cells were mechanically dissociated by gentle pipetting in minimum Eagle's medium (Invitrogen) and seeded onto poly-L-lysine-coated 24-well plates (Nunc) at a density of 2 x 105 cells/well. After a 3-h incubation, the culture medium was replaced with B27 Neurobasal medium (Invitrogen) supplemented with 200 mM glutamine, 10 mM glutamate, and penicillin/streptomycin. Cells were cultured in an incubation chamber gassed with 5% CO2, 95% air at 37 °C. For transfection, cells 7 days in vitro were transfected with LF2000 (5 µl) and 2 µg of DNAs for 5 h in Opti-MEM (Invitrogen). Cells were washed with PBS once and fresh B27 medium once and were incubated for another 72 h. Immunostaining of an astrocyte marker (glial fibrillary acidic protein) indicated that the neuronal cultures were free of astrocytes (data not shown).

Immunofluorescence—Seventy two hours after transfection, cells grown on glass coverslips were fixed with 4% paraformaldehyde, 4% sucrose in PBS for 30 min at room temperature and permeabilized by 0.05% Nonidet P-40 in PBS at room temperature three times for 20 min each time. After a 1-h blocking in 2% PBS, bovine serum albumin, 2% normal goat serum, cells were stained with the desired primary antibody reconstituted in PBS, 2% goat serum at 4 °C for 14–16 h. Dilution of the anti-MAP2 antibody (Chemicon International, Temecula, CA) was 1:500. The anti-Htt antibody was raised against the first 17 amino acids of human Htt plus two glutamine residues and was utilized at a dilution of 1:500. After extensive washing, the slides were incubated with the corresponding secondary antibody conjugated with Alexa Red (1:500; Molecular Probes, Leiden, The Netherlands) for 1 h at room temperature, followed by washing, and analysis with the aid of a laser confocal microscope (Bio-Rad, MRC-1000).

Measurement of Mutant Htt Aggregate Formation—For measuring aggregate formation, cells were transfected with the indicated pcDNA3.1-Htt-(Q)n-hrGFP construct and incubated for 3 days. Cells with green fluorescence were identified and counted under a confocal laser fluorescence microscope (MRC-1000, Bio-Rad). Percentages of cells containing visible fluorescent aggregates were quantified. Approximately 100 transfected cells were counted. Data points represent the mean ± S.E. of at least three independent experiments.

Luciferase Assays—Seventy two hours post-transfection, cells were harvested and lysed in the Cell Culture Lysis Buffer of the Dualluciferase Reporter Assay System (Promega). Lysates were then centrifuged at 1500 rpm for 5 min to remove insoluble materials. Luciferase activities of 10 µl of lysate were determined following the manufacturer's protocol using a TD-20/20 Luminometer (Promega). Results were obtained by using at least two different preparations of plasmids. The protein concentrations of lysates were determined by the Bradford analysis (37), and the luciferase activities were normalized to the amount of proteins in the lysate. At least three independent transfections were performed for each experiment.

Galactosidase Detection—To determine the expression of {beta}-galactosidase in the transfected cells, cells were first lysed in the Cell Culture Lysis Buffer (Promega). After low speed centrifugation, the supernatant was collected to determine the activity of {beta}-galactosidase using o-nitrophenyl-{beta}-D-galactopyranoside as the substrate by measuring the absorbance of the product at 420 nm with a spectrophotometer. The {beta}-galactosidase activity was linear with time for A420 values up to 1.

Western Blot Analysis—Equal amounts of protein were separated by SDS-PAGE by using 10% polyacrylamide gels according to the method of Laemmli (38). The resolved proteins were electroblotted onto Immobilon polyvinylidene difluoride membranes (Millipore, Bedford, MA). Membranes were blocked with 5% skim milk in PBS and incubated with an anti-CBP antibody (1:1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), anti-CREB antibody (1:2000 dilution; UpState, Charlottesville, VA), anti-phosphorylated CREB antibody (1:1000 dilution; UpState), or anti-lamin antibody (1:2000 dilution; Santa Cruz Biotechnology) at 4 °C overnight followed by the corresponding secondary antibody for 1 h at room temperature. Immunoreactive bands were detected by enhanced chemiluminescence (Pierce) and recorded using Kodak XAR-5 films.

Flow Cytometry and Cell Sorting—Cells were harvested by centrifugation, resuspended in PBS to a final density of 5 x 106 cells/ml, and filtered through a nylon membrane to remove cell aggregates. Flow cytometry and sorting of hrGFP-positive cells were performed using a FACSVantage (BD Biosciences) with a 530 ± 15-nm bandpass filter as they traversed the beam of an argon ion laser (488 nm, 100 milliwatts). Data acquisition and analysis were performed with Cellquest software (BD Biosciences). Sorted cells were harvested into tubes for further isolation of total RNA or protein.

RNA Isolation and Quantitative Real Time PCR—Total RNA was isolated using the TriReagent kit (Molecular Research Center, Cincinnati, OH), treated with RNase-free DNase (RQ1; Promega) to remove the potential contamination of genomic DNA, and transcribed into cDNA using Superscript* II reverse transcriptase. Real time quantitative PCR was performed using the TaqMan kit (PE Applied Biosystems, Foster City, CA) on a TaqMan ABI 7700 Sequence Detection System (PE Applied Biosystems) using a heat-activated TaqDNA polymerase (Amplitaq Gold; PE Applied Biosystems). The PCR mixtures were incubated at 50 °C for 2 min and 95 °C for 10 min, and then 40 PCR cycles were conducted (95 °C for 15 s and 65 °C for 1 min). The sequences of primers are listed below: for A2A-R (the target gene), 5'-TCGCCTGTTTTGTCCTGC-3' and 5'-ATTCATGGGCACCACGTCC-TCGAAC-3'; for Bcl-2 (the target gene), 5'-CATGCTGGGGCCGTACAG-3' and 5'-GAACCGGCACCTGCACAC-3'; and for GAPDH (the reference gene), 5'-TATCCGTTGTGGATCTGACAT-3' and 5'ACA-ACCTGGTCCTCAGTGTA-3'. Independent reverse transcription-PCRs were performed using the same cDNA for both the target gene and reference gene. A melting curve was created at the end of the PCR cycle to confirm that a single product was amplified. Data were analyzed by the ABI 7700 operating software to determine the threshold cycle (CT) above the background for each reaction. The relative transcript amount of the target gene, which was calculated using standard curves of serial RNA dilutions, was normalized to that of GAPDH of the same RNA.

Nuclear Extract Preparation—Cells (5 x 106) were washed with 1x PBS (137 mM NaCl, 2.6 mM KCl, 9.5 mM Na2HPO4, and 1.4 mM KH2PO4 (pH 7.4)), suspended in 500 µl of buffer A (10 mM Hepes (pH 8), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 1 mM Na3VO4, 20 mM NaF, 100 nM okadaic acid, and 0.5% Nonidet P-40), and homogenized by Dounce strokes five times in buffer A. After centrifugation at 2000 rpm for 1 min at 4 °C, the supernatant was collected into new tubes and then centrifuged at 5000 rpm for 5 min at 4 °C. The pellet was then resuspended in 200 µl of buffer B (20 mM Hepes (pH 8), 425 mM NaCl, 1.5 mM MgCl, 0.2 mM EDTA, 0.5 mM dithiothreitol, 80 mg/ml phenylmethylsulfonyl fluoride, 1 mM NaVO4, 20 mM NaF, 100 nM okadaic acid, and 25% glycerol) and incubated for 60 min on ice. Nuclear extracts were next collected by centrifugation at 14,500 rpm for 5 min at 4 °C. The protein concentration of the nuclear extracts in the supernatant was measured with the Bio-Rad protein assay reagent.

Electrophoretic Gel Mobility Shift Assay (EMSA)—The double-stranded DNA fragment comprising the core promoter region of the A2A-R gene (-250 to -145) was generated by excising the indicated DNA fragment from the core-promoter construct (pGL2-[-250/-145]) with BstXI and NheI. DNA fragments harboring the desired mutations at the CRE sites were prepared by using PCR as described above. The control DNA fragment (B-120) of 120 bp was generated by excising pGL2-Basic with EcoRI and XcmI. The excised DNA fragments were separated and purified from 1.2% DNA gels. The double-stranded oligonucleotides of 20 bp containing the wild type (WT) or mutated CRE were prepared by annealing two complementary oligonucleotides under the following conditions: 88 °C for 2 min, 65 °C for 10 min, 37 °C for 10 min, 25 °C for 25 min, and 4 °C for 15 min. The sequences of oligonucleotides are listed below: for wild type, 5'-ACTCTCTCTTTCCAGGTATC-3' and 5'-GATACCTGGAAAGAGAGAGT-3'; for M4, 5'-ACTCTCTCTTGACCTGTATC-3' and 5'-GATACAGGTCAAGAGAGAGT-3'; and for M6, 5'-ACTCTCTCTTGAACTTTATC-3' and 5-GATAAAGTTCAAGAGAGAGT-3'. The sequence of an irrelevant 20-bp control oligonucleotide (B-20) is 5'-TACCGAGCTCTTACGCGTGC-3' and 5'-CGTGCGCATTCTCGAGCCAT-3'. The labeled probes (2.5 x 104 cpm) were incubated with nuclear extract (5 µg) and poly(dI)-poly(dC) (2 µg) in a reaction buffer (3 mM Hepes (pH 7.9), 3.2 mM KCl, 0.02 mM EDTA, 0.1 mM dithiothreitol, 0.16 mM MgCl2, and 10% glycerol). Reactions were incubated on ice for 10 min and loaded onto an 8 or a 6% native polyacrylamide gel for the 105- or 20-bp DNA probe, respectively. The gel was run in 0.25x TBE buffer (89 mM boric acid, 2.5 mM EDTA, and 89 mM Tris (pH 8)) at 4 °C for 2.5–4 h at 120–150 V, dried, and autoradiographed. For the supershift analyses, 1 or 2 µg of an anti-CREB antibody (UpState, Charlottesville, VA) or a nonrelated AC6D antibody (39) was incubated with 5 µg of nuclear extract (1 h at 4 °C) prior to the addition of the binding buffer and radiolabeled probe, and the same procedures were followed as described above.

To determine whether CREB and CBP exist in the protein-promoter complexes, unlabeled A2AR-250/-145 probes (10 or 20 µg) harboring the wild type or the desired mutated CRE site were incubated with nuclear extract (0.5 mg, respectively) of PC12 cells under the same conditions described above for EMSA. The resultant gels were first stained with Coomassie Blue to identify the location of the proteins-DNA complex. The complex was excised, mixed with 1x SDS sample treatment buffer, boiled for 5 min, and separated on 10% SDS-PAGE. Following electrophoresis, gels were transferred to polyvinylidene difluoride membranes for Western blot analysis using an anti-CREB antibody or an anti-CBP antibody as described above.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
The A2A-R is located in striatal GABAergic neurons, which are markedly degenerated during the progression of HD. Previous studies have reported that the striatal level of the A2A-R is markedly reduced in HD (10, 11). Because A2A-R-related drugs have been proposed for developing treatments for HD (27, 29), we set out to investigate the molecular mechanism underlying the reduction of A2A-R expression in HD. To characterize gene regulation of the A2A-R by mutant Htt, expression constructs were created of mutant Htt (pcDNA3-Htt-(Q)n-hrGFP) that encode chimeras (Htt-(Q)n-hrGFP) containing hrGFP at the C terminus fused to a fragment of Htt encoded by its exon-1 and the indicated number of poly(Q) repeats. Transient expression of Htt-(Q)109-hrGFP in primary striatal neuronal culture for 3 days produced Htt aggregates that could readily be observed and quantified by using confocal microscopy (Table I; Fig. S-1 of the Supplemental Material). In primary striatal neurons, more than half (56.9% ± 4.2%) of the cells transfected with Htt-(Q)109-hrGFP contained neuronal intranuclear inclusions, whereas those transfected with Htt-(Q)25-hrGFP showed only dispersed hrGFP in the cytoplasm. Similarly, transient expression of mutant Htt with expanded poly(Q) led to the formation of aggregates in PC12 cells (Table I; Fig. S-2 of the Supplemental Material). With 48 or 73 copies of poly(Q), ~15% of the transfected PC12 cells harbored Htt aggregates in the cytoplasm, whereas no neuronal intranuclear inclusions were detected. Further expansion of poly(Q) to as many as 109 copies led to a more severe aggregation of mutant Htt in both the cytoplasm and nucleus (Fig. S-2, E and F). The aggregation of mutant Htt was verified by colocalization of hrGFP and Htt as shown in Fig. S-2H. Most intriguingly, stimulation of the A2A-R using an A2A-R-selective agonist (CGS21680 CGS) significantly reduced the formation of neuronal intranuclear inclusions and cytoplasmic Htt aggregations (Table I). Because a reduction in the size or number of Htt aggregates has been observed concurrent with improvements in major symptoms of HD and/or reduced toxicity caused by poly(Q)-associated Htt (7, 40–42), suppression of Htt aggregation by the A2A-R might lead to ameliorated symptoms of HD, such as abnormal gene regulation.


View this table:
[in this window]
[in a new window]
  		TABLE I
Intracellular localization of Htt aggregates in PC12 cells and primary striatal neurons PC12 cells and primary striatal neurons were transfected with the mutant Htt construct with the indicated number of poly(Q) (Htt-(Q)n-hrGFP). Forty eight hours post-transfection, PC12 cells were treated with or without an A2A-R agonist (CGS, 1 µM) for 24 h as indicated. Percentages of cells containing Htt aggregates in the cytoplasm and/or in nuclei were quantified using fluorescence microscopy. For each experiment, approximately 100 transfected cells were counted. Data points represent the mean ± S.E. of at least three independent experiments.

 
We first determined whether mutant Htt with 109Q affected the expression of the endogenous A2A-R in PC12 cells and in primary striatal neurons. Cells were transiently transfected with Htt-(Q)25-hrGFP or Htt-(Q)109-hrGFP as indicated. The hrGFP-positive cells were identified and harvested using a flow cytometer to collect RNA. Expression levels of the endogenous A2A-R in the presence of mutant Htt with either 25Q or 109Q were determined using a quantitative PCR method. As shown in Fig. 1A, expression of mutant Htt with expanded poly(Q) markedly reduced the expression of the endogenous A2A-R in both primary striatal neurons and PC12 cells. The suppressive effect of mutant Htt was selective because no detectable effect on endogenous bcl2 expression was found (Fig. 1B).



View larger version (30K):
[in this window]
[in a new window]
  		FIG. 1.
Expression of mutant Htt with expanded poly(Q) reduced the expression of endogenous A2A-R, but not that of Bcl-2, in PC12 cells and primary striatal neurons. PC12 cells and primary striatal neurons were transfected with pcDNA3.1-(Q)25-Htt-hrGFP or pcDNA3.1-(Q)109-Htt-hrGFP as indicated. Seventy two hours post-transfection, cells were harvested and sorted by the expression of hrGFP. Total RNA was collected from the sorted cells and was used for the quantitative PCR analysis. Expression levels of the A2A-R (A) or Bcl-2 (B) were normalized to that of the GAPDH. Data are expressed as the mean ± S.E. of three independent experiments. **, specific comparison between cells transfected with pcDNA3.1-(Q)109-Htt-hrGFP and cells transfected with pcDNA3.1-(Q)25-Htt-hrGFP (p < 0.001; one-way analysis of variance).

 
To determine whether mutant Htt affects expression of the A2A-R gene by regulating its promoter, the pGL2-(-1997/-145) construct, which contains two promoters of the rat A2A-R gene (18), and the expression construct encoding mutant Htt with the indicated number of CAG repeats (Q) were cotransfected into PC12 and primary striatal neurons. As shown in Fig. 2, marked repression of the A2A-R promoter was observed in the presence of 109Q-Htt in both cell types tested. To locate the DNA region responsible for the above suppressive effect, a series of 5'-deleted A2A-R promoter constructs was transfected into PC12 cells along with mutant Htt harboring 25Q or 109Q. The suppressive effect of 109Q-Htt persisted when the A2A-R gene fragment was 5'-deleted to -250 bp where the major core promoter region resides (Fig. 3A) (18). Thus, poly(Q)-expanded Htt inhibited A2A-R expression by suppressing its core promoter region (18). Consistently, the proteins-A2A-R promoter complex from cells expressing 109Q-Htt migrated faster than those from cells containing 25Q-Htt, indicating that specific binding of nuclear proteins to the core promoter region of the A2A-R gene was altered in the presence of 109Q-Htt when compared with 25Q-Htt (Fig. 3B). Note that the proteins-A2A-R promoter complex from nontransfected PC12, which expressed no mutant Htt, migrated to the same position as that from PC12 cells expressing Htt with 25Q (Fig. S-3; Supplemental Material). Overexpression of the Htt fragment by itself therefore did not alter formation of the proteins-A2A-R promoter complex. Most importantly, such a suppressive effect of poly(Q)-expanded Htt was also observed on three other promoters (p21, SV40, and TK) but not on the cytomegalovirus promoter (Table II), further supporting the hypothesis that alteration of gene regulation by mutant Htt is specific.



View larger version (24K):
[in this window]
[in a new window]
  		FIG. 2.
Expression of mutant Htt with expanded poly(Q) suppressed the A2A-R promoter in PC12 cells and in primary striatal neurons. The indicated Htt construct with the desired number of poly(Q) was cotransfected with a previously characterized A2A-R promoter construct that contains a 1.8-kb 5'-flanking fragment of the rat A2A-R gene (pGL2-(-1997/-145)) (18) into PC12 cells or primary striatal neurons for 72 h. Luciferase activity of each construct was measured and normalized to the protein content. Values are expressed as percentages of the promoter activity in the presence of pcDNA3.1-(Q)25-Htt-hrGFP in the indicated cell type and represent the mean ± S.E. of at least three independent experiments. **, specific comparison between cells transfected with the indicated construct and cells transfected with pcDNA3.1-(Q)25-Htt-hrGFP (p < 0.001; one-way analysis of variance).

 



View larger version (26K):
[in this window]
[in a new window]
  		FIG. 3.
Expression of poly(Q)-expanded Htt suppressed the core promoter activity and altered the binding of nuclear proteins to the core promoter of the rat A2A-R gene in PC12 cells. A, indicated Htt construct with the desired number of poly(Q) was cotransfected with a series of previously characterized A2A-R promoter constructs that contain the indicated 5'-flanking fragment (18) along with pGL2-basic, which lacks promoter activity, into PC12 cells for 72 h. Values are expressed as percentages of the promoter activity of pGL2-(-1997/-145) in the presence of pcDNA3.1-(Q)25-Htt-hrGFP and represent the mean ± S.E. of at least three determinations. **, specific comparison between cells transfected with pcDNA3.1-(Q)109-Htt-hrGFP and cells transfected with pcDNA3.1-(Q)25-Htt-hrGFP (p < 0.001; one-way analysis of variance). Luciferase (LUC), a coding region of luciferase. B, PC12 cells were transfected with pcDNA3.1-(Q)25-Htt-hrGFP or pcDNA3.1-(Q)109-Htt-hrGFP. Forty eight hours post-transfection, cells were treated with or without CGS (1 µM) for an additional 24 h. GFP-positive cells were then harvested by fluorescence-activated cell sorter to prepare the corresponding nuclear extract (NE). Double-stranded 105-bp DNA containing the core promoter region of the A2A-R gene (A2AR-250/-145) was 32P-labeled and incubated with nuclear extract (5 µg) prepared from PC12 cells expressing (Q)25-Htt-hrGFP or (Q)109-Htt-hrGFP as indicated. Formation of DNA-protein complexes competed with the indicated double-stranded A2AR-250/-145 or the control 120-bp DNA (B-120) in a 50-fold molar excess. Reaction products were separated in 8% polyacrylamide gels by electrophoresis. This is one representative result of three independent experiments performed.

 


View this table:
[in this window]
[in a new window]
  		TABLE II
Effect of mutant Htt with expanded poly(Q) on four different promoters PC12 cells and primary striatal neurons were transfected with the indicated promoter construct plus pcDNA3.1-(Q)25-Htt-hrGFP or pcDNA3.1-(Q)109-Htt-hrGFP as indicated for 72 h. Reporter activity of each construct was measured and normalized to the protein content. Values are expressed as percentages of the promoter activity in the presence of pcDNA3.1-(Q)25-Htt-hrGFP, and represent the mean ± S.E. of at least three independent experiments.

 
Another very interesting observation was that stimulation of the A2A-R in the presence of 109Q-Htt by CGS restored the proper protein-binding pattern in the EMSA to that with 25Q-Htt (Fig. 3B). Activation of the A2A-R thus might protect inhibition of the A2A-R promoter from poly(Q)-expanded Htt. In agreement with the above observation, CGS treatment elevated the reduced promoter activity by 109Q-Htt (Fig. 4A). Moreover, pretreating cells with an A2A-R-selective antagonist, 8-(3-chlorostyryl) caffeine (CSC), markedly reduced the effect of CGS (Fig. 4A). The elevating effect of CGS therefore appeared to be specifically mediated by the A2A-R. Because stimulation of the A2A-R has been shown to cause a transient increase in cAMP in PC12 cells (43), we next examined whether the cAMP/PKA pathway plays an important role in the A2A-R reversing effect. As shown in Fig. 4A, direct stimulation of adenylyl cyclase using forskolin (FK), which subsequently activates the cAMP/PKA pathway, also significantly reversed the reduction in A2A-R promoter activity by 109Q-Htt. Treatment with two selective PKA inhibitors (H-89 and KT5720) effectively blocked the reversing effect of CGS and FK. In addition, CGS exerted no reversing effect on the suppressed A2A-R promoter activity by 109Q-Htt in a PKA-deficient PC12 variant (A123, Fig. 4B). PKA therefore is essential for the rescuing effect of A2A-Rs.



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 4.
Stimulation of the A2A-R selectively reversed the suppression of the A2A-R promoter by poly(Q)-expanded Htt in PC12 cells. A, indicated Htt construct was cotransfected with an A2A-R promoter construct (pGL2-(-1997/-145)) into PC12 cells for 48 h. Cells were then treated with the indicated inhibitor (10 µM CSC, 10 µM H89, or 1 µM KT5720) for 30 min, followed by a 24-h incubation with CGS (1 µM) or forskolin (10 µM). B, the indicated Htt construct was cotransfected with an A2A-R promoter construct (pGL2-(-1997/-145)) into a PKA-deficient PC12 cell variant (A123) for 48 h. Cells were then treated with CGS (1 µM) or forskolin (10 µM) for 24 h. Luciferase activity of each construct was measured and normalized to the protein content. Values are expressed as percentages of the promoter activity of pGL2-(-1997/-145) in the presence of pcDNA3.1-(Q)25-Htt-hrGFP under control conditions (no treatment) and represent the mean ± S.E. of at least three determinations. a, specific comparison between the indicated group and that transfected with pcDNA3.1-(Q)109-Htt-hrGFP with no treatment (p < 0.001; one-way analysis of variance). b, specific comparison between control and inhibitor-treated cells in each group (p < 0.001; one-way analysis of variance).

 
Because CREB has been implicated in the action of mutant Htt (2, 44), and because we earlier demonstrated that stimulation of the A2A-R led to activation of CREB via PKA (10), we next investigated the involvement of CREB in the promoter activity of the A2A-R-reversing effect. In PC12 cells, expression of two dominant negative CREB mutants (CREBm1 and CREB-R287L) was shown previously to significantly reduce the activity of CREB upon A2A-R stimulation (6). Expression of either CREBm1 or CREB-R287L effectively reduced the A2A-R-reversed suppression of the A2A-R promoter caused by 109Q-Htt (Fig. 5A). Consistently, overexpression of a constitutively active CREB mutant (VP16-CREB, Fig. 5B), but not VP16 alone, markedly elevated A2A-R promoter activity. CREB thus appears to play a critical role in the action of A2A-R. The observation that VP16-CREB exhibited a much more significant effect on elevating the activity of the A2A-R promoter than that of A2A-R stimulation was because of the latter only causing transient elevation in cAMP/CREB (43).



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 5.
CREB played a critical role in mediating the effect of A2A-R on suppression of the A2A-R promoter by poly(Q)-expanded Htt. A, PC12 cells were transiently transfected with the indicated mutant Htt construct, an A2A-R promoter construct (pGL2-(-1997/-145)), and a construct encoding a negative CREB mutant (CREBm1 or CREBR287), or an empty vector in the molar amounts of 7, 7, and 1, respectively, for 48 h. Cells were then treated with a 24-h incubation with or without CGS (1 µM). B, PC12 cells were transiently transfected with the indicated mutant Htt construct, an A2A-R promoter construct (pGL2-(-1997/-145)), and a construct encoding a constitutively active CREB mutant (VP16-CREB) or a control vector (VP16) in the molar amounts of 7, 7, and 1, respectively, for 72 h. Luciferase activity of each construct was measured and normalized to the protein content. Values are expressed as percentages of the promoter activity of pGL2-(-1997/-145) in the presence of pcDNA3.1-(Q)25-Htt-hrGFP under control (no treatment) conditions, and represent the mean ± S.E. of at least three determinations. a, specific comparison between the indicated group and that transfected with pcDNA3.1-(Q)109-Htt-hrGFP with no additional treatment (p < 0.001; one-way analysis of variance). b, specific comparison between the control vector and the CREB mutant-expressing cells in each group (p < 0.001; two-way analysis of variance).

 
Most interestingly, an atypical CRE site (TCCAGG, -246 to -241) (45) is located in the core promoter region of the A2A-R gene (Fig. 6A). To determine whether CREB binds to the core promoter region of the A2A-R gene, we carried out combined EMSA and Western blot analyses. We first performed EMSA by using four A2A-R core promoter probes that contain wild type or distinct mutations in the CRE region (Table III). Mutations at only two residues (designated M2) was shown previously to retain the binding activity of CREB, whereas the M6 mutant completely lost its CREB binding ability (46). The proteins-DNA complexes were first isolated from nondenaturing gels in the EMSA and then were subjected to separation by SDS-PAGE, followed by Western blot analyses. The presence of CREB and a CREB-binding protein (CBP) in the protein complex bound to the wild type and M2 core promoters of the A2A-R gene is clearly demonstrated in Fig. 6B. In contrast, CREB and CBP were not detected in the proteins-M4 and proteins-M6 complexes. To verify the direct involvement of CREB in regulating the A2A-R promoter, an anti-CREB antibody or irrelevant serum (39) was incubated with nuclear extract collected from the PC12 cells expressing Htt-(Q)25-hrGFP or Htt-(Q)109-hrGFP prior to incubation with a labeled A2A-R core promoter probe (A2AR-250/-145) for the EMSA. As shown in Fig. 6C, adding the anti-CREB antibody, but not an irrelevant antibody (AC6D), created a supershift in the EMSA of control cells expressing Htt-(Q)25-hrGFP. In contrast, no supershift in the EMSA complex was found in cells expressing Htt-(Q)109-hrGFP, suggesting that CREB did not bind to the core promoter of the A2A-R gene in the presence of poly(Q)-expanded Htt. These results support the hypothesis that a functional CRE site is located in the core promoter region of the rat A2A-R gene and is involved in mutant Htt-evoked suppression of the A2A-R gene. Moreover, the anti-CREB antibody caused a supershift in the EMSA of cells expressing Htt-(Q)109-hrGFP treated with CGS (Fig. 6C), demonstrating that A2A-R stimulation restored CREB binding to the core promoter of the A2A-R gene, as well as by elevating the suppressed A2A-R promoter activity by poly(Q)-expanded Htt (Fig. 4A).



View larger version (34K):
[in this window]
[in a new window]
  		FIG. 6.
CREB directly bound to the core promoter of the A2A-R gene. A, atypical CRE site (underlined) is located at the core promoter region of the A2A-R. B, a combination of EMSA and Western blot analyses was performed as follows: 10 µg of the unlabeled A2AR (-250/-145) probes containing the wild type (WT) or the mutated CRE site (M2, M4, or M6) as indicated was first incubated with 0.5 mg of nuclear extract of PC12 cells and subjected to EMSA electrophoresis. Gels were stained with Coomassie Blue to identify the location of the proteins-DNA complex. The complex was then excised and separated by SDS-PAGE, followed by Western blot analyses using an anti-CREB and an anti-CBP antibody. C, supershift analyses of the A2A-R core promoter complex. NE (2.5 µg) was prepared from PC12 cells transfected with pcDNA3.1-(Q)25-Htt-hrGFP or pcDNA3.1-(Q)109-Htt-hrGFP as described in the legend of Fig. 3B, and NE was preincubated for 1 h with anti-CREB or an irrelevant anti-AC6D (39) antibody, followed by incubation with double-stranded, 32P-labeled 105-bp DNA containing the core promoter region of the A2A-R gene (A2AR-250/-145) for EMSA. Reaction products were separated in 8% polyacrylamide gels by electrophoresis. This is one representative result of three independent experiments performed. Arrowhead indicates the supershifted bands. Arrows mark the protein-A2A-R promoter complexes.

 


View this table:
[in this window]
[in a new window]
  		TABLE III
Mutations in the atypical CRE site at the rat A2A-R core promoter The mutated residues are underlined.

 
To further verify that the CRE site is important for A2A-R core promoter activity, we first performed EMSA using three A2A-R core promoter probes that contain distinct mutations in the CRE region (Table III). Mutations at only two residues (M2), which were shown not to affect the binding activity of CREB (46), did not change the EMSA profile (Fig. 7A). In contrast, the M6 mutant that lacks CREB binding activity (46) caused a downshift of the bands in the presence of 25Q-Htt (Fig. 7A). The EMSA pattern of the M4 mutant that harbors mutations at four residues was very similar to that of M6, suggesting that M4 is also a null CRE site. These findings are in line with the combined EMSA/Western analyses that showed that CREB and CBP did not bind to the M4 or M6 probes (Fig. 6B). Downshifts of the proteins-M4 and proteins-M6 complexes in the presence of 25Q-Htt reflect the loss of CREB/CBP binding to the core promoter of the A2A-R gene. Consistently, in the presence of 109Q-Htt, none of those CRE mutants exhibited altered EMSA profiles. Therefore, CREB did not bind the core promoter of the A2A-R gene in the presence of poly(Q)-expanded Htt.



View larger version (30K):
[in this window]
[in a new window]
  		FIG. 7.
The functional CRE site was critical for A2A-R promoter activity and contributed to suppression of the A2A-R gene by Htt with expanded poly(Q). A, nuclear extract (2.5 µg) prepared from cells expressing (Q)25-Htt-hrGFP or (Q)109-Htt-hrGFP as described in Fig. 6 was reacted with a 32P-labeled A2AR-250/-145 oligonucleotide probe containing the wild type (WT) or the mutated CRE site (M2, M4, or M6) as indicated. Reaction products were separated in 8% polyacrylamide gels by electrophoresis. This is one representative result of three independent experiments performed. B, the indicated Htt construct with the desired number of poly(Q) was cotransfected with these four A2A-R core promoter constructs (-250/-145) that contain the WT or the mutated CRE site (M2, M4, or M6) as indicated into PC12 cells for 72 h. Forty eight hours post-transfection, cells were treated with or without an the A2A-R agonist (CGS, 1 µM) or forskolin (FK, 10 µM) for 24 h. Values are expressed as percentages of the promoter activity of wild type pGL2-(-250/-145) in the presence of pcDNA3.1-(Q)25-Htt-hrGFP and represent the mean ± S.E. of at least three determinations. a, specific comparison between the indicated CRE mutant and the WT in the presence of 25Q-Htt. b, specific comparison between control, nontreated cells transfected with pcDNA3.1-(Q)109-Htt-hrGFP and those with pcDNA3.1-(Q)25-Htt-hrGFP in the corresponding group. c, specific comparison between cells treated with or without the indicated drug in the presence of 109Q-Htt in each corresponding group. d, specific comparison of the suppression of A2A-R promoter activity by109Q-expanded Htt mediated between WT and the indicated CRE mutant. e, specific comparison of the CGS or forskolin-rescuing effect between WT and the indicated CRE mutant (p < 0.05; one-way analysis of variance).

 
Consistent with the observation based on the EMSAs of cells expressing Htt with the normal repeat number of poly(Q) (25), promoter constructs containing inactivated CRE sites (M4 or M6) exhibited markedly reduced promoter activities compared with those containing a functional CRE site (WT or M2; Fig. 7B). Binding of CREB therefore plays an important role in the basal promoter activity of the A2A-R gene. Although expression of 109Q-Htt caused a suppression of all A2A-R promoters tested, the inhibition was much less severe for the M4 and M6 promoters. The rescue effect of A2A-R stimulation on inhibition of the A2A-R promoter by 109Q-Htt was also significantly reduced in promoters containing null CRE sites (M4 or M6). Likewise, the marked enhancing effect of forskolin on wild type and M2 promoters in the presence of expanded poly(Q) was greatly reduced in the CRE-null mutant promoters (M4 and M6). The CRE site therefore was significantly involved in regulation of the A2A-R promoter by mutant Htt with expanded poly(Q). Note that in the presence of the null CRE site, 109Q-Htt retained the ability to slightly suppress A2A-R promoter activity which could also be moderately rescued by A2A-R stimulation (Fig. 7B). Other transcription factor(s), in addition to CREB, thus might also contribute to the regulation of A2A-R promoter by poly(Q)-expanded Htt.

Because the A2A-R core promoter probe (105 bp, A2AR-250/-145) is relatively large and might bind proteins other than CREB, we next utilized shorter oligonucleotides probes (20 bp, A2AR-256/-237) that contain wild type or the null CRE site (M4 or M6 mutants) to verify the importance of CREB. When the radiolabeled probe harboring the wild type CRE site was incubated with nuclear extracts from nontransfected PC12 cells or cells expressing Htt with 25Q, a prominent DNA-protein complex was observed. Such a complex disappeared in the presence of an excess amount of the unlabeled wild type probe (A2AR-256/-237), but not an irrelevant 20-bp probe (B-20; Fig. 8A). Adding the anti-CREB antibody, but not an irrelevant antibody (AC6D), created a supershift in the EMSA of cells expressing Htt-(Q)25-hrGFP. These data further support the hypothesis that CREB recognizes the atypical CRE site of the A2A-R core promoter. No DNA-protein complex was detected from cells expressing 109Q-Htt, demonstrating that CREB did not bind to the atypical CRE site in the presence of poly(Q)-expanded Htt (Fig. 8A). Moreover, no DNA-protein complex was found when the DNA probe contained a mutated CRE site (M4 or M6; Fig. 8B). Collectively, the above EMSA analyses using oligonucleotide probes containing only the atypical CRE site strengthen our hypothesis that binding of CREB to this atypical CRE site was altered by the expression of poly(Q)-expanded Htt and might be involved in mutant Htt-evoked suppression of the A2A-R gene.



View larger version (43K):
[in this window]
[in a new window]
  		FIG. 8.
Expression of poly(Q)-expanded Htt interfered with the binding of CREB to the atypical CRE site of the A2A-R gene. PC12 cells were transfected with pcDNA3.1-(Q)25-Htt-hrGFP or pcDNA3.1-(Q)109-Htt-hrGFP. Seventy two hours post-transfection, GFP-positive cells were harvested by fluorescence-activated cell sorter to prepare the corresponding NE. A, double-stranded oligonucleotide probe (20 bp; A2AR-256/-237) that harbors the wild type CRE site was 32P-labeled and incubated with nuclear extract (5 µg) prepared from nontransfected PC12 cells (PC) or transfected PC12 cells expressing (Q)25-Htt-hrGFP or (Q)109-Htt-hrGFP as indicated. Formation of DNA-protein complexes competed with the indicated double-stranded A2AR-256/-237 or the control 20-bp DNA (B-20) in a 50-fold molar excess. For the supershift analyses, NE (5 µg) prepared from the indicated cells was preincubated for 1 h with an anti-CREB or an irrelevant anti-AC6D (39) antibody, followed by incubation with the 32P-labeled 20-bp DNA probe for EMSA. PC, nontransfected PC12 cells. B, NE (5 µg) prepared from cells expressing (Q)25-Htt-hrGFP or (Q)109-Htt-hrGFP was reacted with a 20-bp 32P-labeled A2AR-250/-230 oligonucleotide probe containing the wild type (WT) or the mutated CRE site (M4 or M6) as indicated. Reaction products were separated in 6% polyacrylamide gels by electrophoresis. This is one representative result of three independent experiments performed. The arrowhead indicates the supershifted bands. Arrows mark the protein/A2A-R promoter complexes.

 
We next determined whether the lack of CREB binding and the suppression of A2A-R promoter activity by poly(Q)-expanded Htt were because of lower amounts of soluble CREB and its cofactor (CBP) using Western blot analyses. As shown in Fig. 9, the levels of soluble CREB in cells expressing 25Q-Htt or 109Q-Htt were very similar. The lack of CREB binding to the A2A-R promoter therefore might not have been due to the sequestration of CREB by Htt aggregates but rather might have been caused by conformational inactivation as reported for other transcription factors by toxic Htt (47). Most interestingly, the amounts of activated CREB, determined by assessing the phosphorylation level at Ser133, and the levels of CBP were reduced in cells expressing poly(Q)-expanded Htt. Both deficiencies should contribute to suppression of the A2A-R gene by poly(Q)-expanded Htt.



View larger version (35K):
[in this window]
[in a new window]
  		FIG. 9.
Mutant Htt with expanded poly(Q) reduced the activation of CREB and the expression of CBP. PC12 cells were transfected with pcDNA3.1-(Q)25-Htt-hrGFP or pcDNA3.1-(Q)109-Htt-hrGFP for 72 h. GFP-positive cells were then harvested by fluorescence-activated cell sorter to prepare the corresponding nuclear extract. A, equal amounts of nuclear protein (15 µg) were loaded into each lane and analyzed by Western blotting using the corresponding antibody. B, an illustration is given of arbitrary values for the protein of interest normalized with the level of lamin from three independent experiments. **, specific comparison between cells transfected with pcDNA3.1-(Q)109-Htt-hrGFP and those with pcDNA3.1-(Q)25-Htt-hrGFP in the corresponding group. (p < 0.001; Student's t test).

 
Alterations in the overall gene expression profiles have been reported in several poly(Q) disease models, including those of HD. Previous studies have suggested that poly(Q)-expanded mutant Htt might disrupt the transcription machinery through sequestration or direct inactivation of important transcription factors (3, 6, 12, 47, 48). Among them, the role of CREB has been suggested to be responsible for one of the earliest dysfunctional events at the transcription level in HD (44). Moreover, disruption of CREB has been shown to cause progressive striatal degeneration similar to that found in HD (49). A reduced level of CBP because of enhanced ubiquitination and degradation of CBP has also been implicated in the suppression of CREB-mediated transcription in poly(Q)-associated neurode-generative diseases (47). Our findings that the levels of activated/phosphorylated CREB and CBP were reduced in the presence of poly(Q)-expanded Htt are consistent with those of previous studies, which support a critical role of CREB in HD. The most important observation was that stimulation of the PKA pathway, which activates CREB by phosphorylation, effectively restored the ability of CREB to bind to the A2A-R core promoter and to mediate the proper promoter activity of the A2A-R gene in the presence of toxic poly(Q)-expanded Htt. Phosphorylation of CREB might restabilize its conformation to allow the proper binding to an atypical CRE site on the A2A-R promoter. Our finding that mutant Htt did not affect the level of CREB is in agreement with a recent report using PC12 cells (44). Nevertheless, in contrast to our findings (Figs. 7 and 9), those authors reported that mutant Htt does not alter the binding activity of CREB. Such a discrepancy appears to have resulted from the distinct CRE sites involved. According to Sugars et al. (44), the binding ability of CREB was assessed based on a consensus CRE site (5'-TGACGTCA-3'), whereas the CRE site (5'-TCCAGG-3') at the A2A-R core promoter studied here was atypical. Ionescu and colleagues (46) demonstrated that this atypical CRE site binds CREB less strongly than does the consensus CRE site. Mutant Htt might functionally destabilize CREB, as has been reported for the TATA box-binding protein (47), thus causing reduced binding affinity toward atypical CRE sites but not to the consensus CRE site. Note that this atypical CRE site varies slightly from another atypical CRE site (5'-TCCAGT-3') previously found to be functional in at least four other promoters (50–52), suggesting that expression of these genes might also be affected in HD.

The A2A-R has been shown to exert a protective effect upon a wide variety of traumas (23–25, 27), and both agonists and antagonists of the A2A-R have been implicated in neuronal degenerative diseases (27, 29, 53). In the present study, we identify an atypical CRE site located in the core promoter of the A2A-R gene that mediates the suppression of the A2A-R gene by poly(Q)-expanded Htt. Several lines of evidence suggest that Htt with expanded poly(Q) prevents the binding of CREB to this atypical CRE site and markedly reduces the expression of the A2A-R in HD. Concurrently, stimulation of the A2A-R reduced Htt aggregation (Table I) and restored its own gene expression by a PKA/CREB-mediated pathway. It is possible that stimulation of various receptors (particularly those in the G protein-coupled receptor superfamily) that enhance the PKA/CREB pathway might normalize the transcriptional dysfunction of the CREB-mediated pathway evoked by toxic Htt. Results of the present study provide important insights into both the understanding of gene regulation of the A2A-R and the potential usage of cAMP-elevating drugs in HD.


   FOOTNOTES
 
* This work was supported by National Science Council Grants NSC93-2321-B-001-012 and NSC93-2320-B001-009, Academia Sinica Grants AS91IBC3PP-B and AS91IMB4PP-B, and the Institute of Biomedical Science, Academia Sinica (CRC grants), Taipei, Taiwan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S-1, S-2, and S-3. Back

To whom correspondence should be addressed: Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan. Tel.: 886-2-26523913; Fax: 886-2-27829143; E-mail: bmychern{at}ibms.sinica.edu.tw.

1 The abbreviations used are: HD, Huntington's disease; Htt, Huntingtin; A2A-R, A2A adenosine receptor; CGS, CGS21680 PKA, protein kinase A; CREB, cAMP-response element-binding protein; GADPH, glyceraldehyde-3-phosphate dehydrogenase; PBS, phosphate-buffered saline; FK, forskolin; DMEM, Dulbecco's modified Eagle's medium; hrGFP, humanized green fluorescent protein; EMSA, electrophoretic gel mobility shift assay; CBP, CREB-binding protein; NE, nuclear extract; WT, wild type. Back


   ACKNOWLEDGMENTS
 
We thank Dan Chamberlin and Christine Hsieh for reading and editing the manuscript and Kuan-Yu Chu for assistance with confocal microscopy.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 

  1. The Huntington's Disease Collaborative Research Group (1993) Cell 72, 971-983[CrossRef][Medline] [Order article via Infotrieve]
  2. Steffan, J. S., Kazantsev, A., Spasic-Boskovic, O., Greenwald, M., Zhu, Y. Z., Gohler, H., Wanker, E. E., Bates, G. P., Housman, D. E., and Thompson, L. M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 6763-6768[Abstract/Free Full Text]
  3. Nucifora, F. C., Jr., Sasaki, M., Peters, M. F., Huang, H., Cooper, J. K., Yamada, M., Takahashi, H., Tsuji, S., Troncoso, J., Dawson, V. L., Dawson, T. M., and Ross, C. A. (2001) Science 291, 2423-2428[Abstract/Free Full Text]
  4. Kegel, K. B., Meloni, A. R., Yi, Y., Kim, Y. J., Doyle, E., Cuiffo, B. G., Sapp, E., Wang, Y., Qin, Z. H., Chen, J. D., Nevins, J. R., Aronin, N., and DiFiglia, M. (2002) J. Biol. Chem. 277, 7466-7476[Abstract/Free Full Text]
  5. Dunah, A. W., Jeong, H., Griffin, A., Kim, Y. M., Standaert, D. G., Hersch, S. M., Mouradian, M. M., Young, A. B., Tanese, N., and Krainc, D. (2002) Science 296, 2238-2243[Abstract/Free Full Text]
  6. Li, S. H., Cheng, A. L., Zhou, H., Lam, S., Rao, M., Li, H., and Li, X. J. (2002) Mol. Cell. Biol. 22, 1277-1287[Abstract/Free Full Text]
  7. Ravikumar, B., Stewart, A., Kita, H., Kato, K., Duden, R., and Rubinsztein, D. C. (2003) Hum. Mol. Genet. 12, 985-994[Abstract/Free Full Text]
  8. Ferrante, R. J., Kubilus, J. K., Lee, J., Ryu, H., Beesen, A., Zucker, B., Smith, K., Kowall, N. W., Ratan, R. R., Luthi-Carter, R., and Hersch, S. M. (2003) J. Neurosci. 23, 9418-9427[Abstract/Free Full Text]
  9. Sipione, S., Rigamonti, D., Valenza, M., Zuccato, C., Conti, L., Pritchard, J., Kooperberg, C., Olson, J. M., and Cattaneo, E. (2002) Hum. Mol. Genet. 11, 1953-1965[Abstract/Free Full Text]
  10. Chan, E. Y., Luthi-Carter, R., Strand, A., Solano, S. M., Hanson, S. A., DeJohn, M. M., Kooperberg, C., Chase, K. O., DiFiglia, M., Young, A. B., Leavitt, B. R., Cha, J. H., Aronin, N., Hayden, M. R., and Olson, J. M. (2002) Hum. Mol. Genet. 11, 1939-1951[Abstract/Free Full Text]
  11. Luthi-Carter, R., Strand, A. D., Hanson, S. A., Kooperberg, C., Schilling, G., La Spada, A. R., Merry, D. E., Young, A. B., Ross, C. A., Borchelt, D. R., and Olson, J. M. (2002) Hum. Mol. Genet. 11, 1927-1937[Abstract/Free Full Text]
  12. Zuccato, C., Tartari, M., Crotti, A., Goffredo, D., Valenza, M., Conti, L., Cataudella, T., Leavitt, B. R., Hayden, M. R., Timmusk, T., Rigamonti, D., and Cattaneo, E. (2003) Nat. Genet. 35, 76-83[CrossRef][Medline] [Order article via Infotrieve]
  13. Yohrling, G. J., Farrell, L. A., Hollenberg, A. N., and Cha, J. H. (2003) Mol. Cell. Neurosci. 23, 28-38[CrossRef][Medline] [Order article via Infotrieve]
  14. Cha, J. H. (2000) Trends Neurosci. 23, 387-392[CrossRef][Medline] [Order article via Infotrieve]
  15. Ferre, S., O'Connor, W. T., Fuxe, K., and Ungerstedt, U. (1993) J. Neurosci. 13, 5402-5406[Abstract]
  16. Weaver, D. R. (1993) Brain Res. Mol. Brain Res. 20, 313-327[Medline] [Order article via Infotrieve]
  17. Glass, M., Dragunow, M., and Faull, R. L. (2000) Neuroscience 97, 505-519[CrossRef][Medline] [Order article via Infotrieve]
  18. Chu, Y. Y., Tu, K. H., Lee, Y. C., Kuo, Z. J., Lai, H. L., and Chern, Y. (1996) DNA Cell Biol. 15, 329-337[Medline] [Order article via Infotrieve]
  19. Lee, Y. C., Chang, C. W., Su, C. W., Lin, T. N., Sun, S. H., Lai, H. L., and Chern, Y. (1999) J. Neurochem. 73, 1790-1798[CrossRef][Medline] [Order article via Infotrieve]
  20. Chern, Y., King, K., Lai, H. L., and Lai, H. T. (1992) Biochem. Biophys. Res. Commun. 185, 304-309[Medline] [Order article via Infotrieve]
  21. Lee, Y. C., Chien, C. L., Sun, C. N., Huang, C. L., Huang, N. K., Chiang, M. C., Lai, H. L., Lin, Y. S., Chou, S. Y., Wang, C. K., Tai, M. H., Liao, W. L., Lin, T. N., Liu, F. C., and Chern, Y. (2003) Eur. J. Neurosci. 18, 1786-1796[CrossRef][Medline] [Order article via Infotrieve]
  22. Lee, Y. C., Lai, H. L., Sun, C. N., Chien, C. L., and Chern, Y. (2003) Brain Res. Mol. Brain Res. 111, 61-73[Medline] [Order article via Infotrieve]
  23. Huang, N. K., Lin, Y. W., Huang, C. L., Messing, R. O., and Chern, Y. (2001) J. Biol. Chem. 276, 13838-13846[Abstract/Free Full Text]
  24. Walker, B. A., Rocchini, C., Boone, R. H., Ip, S., and Jacobson, M. A. (1997) J. Immunol. 158, 2926-2931[Abstract]
  25. Day, Y. J., Huang, L., McDuffie, M. J., Rosin, D. L., Ye, H., Chen, J. F., Schwarzschild, M. A., Fink, J. S., Linden, J., and Okusa, M. D. (2003) J. Clin. Investig. 112, 883-891[CrossRef][Medline] [Order article via Infotrieve]
  26. Day, Y. J., Marshall, M. A., Huang, L., McDuffie, M. J., Okusa, M. D., and Linden, J. (2004) Am. J. Physiol. 286, G285-G293
  27. Popoli, P., Pezzola, A., Reggio, R., Caporali, M. G., and Scotti de Carolis, A. (1994) Eur. J. Pharmacol. 257, R5-R6[CrossRef][Medline] [Order article via Infotrieve]
  28. Popoli, P., Pintor, A., Domenici, M. R., Frank, C., Tebano, M. T., Pezzola, A., Scarchilli, L., Quarta, D., Reggio, R., Malchiodi-Albedi, F., Falchi, M., and Massotti, M. (2002) J. Neurosci. 22, 1967-1975[Abstract/Free Full Text]
  29. Blum, D., Galas, M. C., Pintor, A., Brouillet, E., Ledent, C., Muller, C. E., Bantubungi, K., Galluzzo, M., Gall, D., Cuvelier, L., Rolland, A. S., Popoli, P., and Schiffmann, S. N. (2003) J. Neurosci. 23, 5361-5369[Abstract/Free Full Text]
  30. Fink, J. S., Kalda, A., Ryu, H., Stack, E. C., Schwarzschild, M. A., Chen, J. F., and Ferrante, R. J. (2004) J. Neurochem. 88, 538-544[CrossRef][Medline] [Order article via Infotrieve]
  31. Varani, K., Abbracchio, M. P., Cannella, M., Cislaghi, G., Giallonardo, P., Mariotti, C., Cattabriga, E., Cattabeni, F., Borea, P. A., Squitieri, F., and Cattaneo, E. (2003) FASEB J. 17, 2148-2150[Abstract/Free Full Text]
  32. Varani, K., Rigamonti, D., Sipione, S., Camurri, A., Borea, P. A., Cattabeni, F., Abbracchio, M. P., and Cattaneo, E. (2001) FASEB J. 15, 1245-1247[Free Full Text]
  33. Wyttenbach, A., Swartz, J., Kita, H., Thykjaer, T., Carmichael, J., Bradley, J., Brown, R., Maxwell, M., Schapira, A., Orntoft, T. F., Kato, K., and Rubinsztein, D. C. (2001) Hum. Mol. Genet. 10, 1829-1845[Abstract/Free Full Text]
  34. Mangiarini, L., Sathasivam, K., Seller, M., Cozens, B., Harper, A., Hetherington, C., Lawton, M., Trottier, Y., Lehrach, H., Davies, S. W., and Bates, G. P. (1996) Cell 87, 493-506[CrossRef][Medline] [Order article via Infotrieve]
  35. Chen, W., Yu, Y. L., Lee, S. F., Chiang, Y. J., Chao, J. R., Huang, J. H., Chiong, J. H., Huang, C. J., Lai, M. Z., Yang-Yen, H. F., and Yen, J. J. (2001) Mol. Cell. Biol. 21, 4636-4646[Abstract/Free Full Text]
  36. Ginty, D. D., Glowacka, D., DeFranco, C., and Wagner, J. A. (1991) J. Biol. Chem. 266, 15325-15333[Abstract/Free Full Text]
  37. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve]
  38. Laemmli, U. K. (1970) Nature 227, 680-685[CrossRef][Medline] [Order article via Infotrieve]
  39. Lai, H. L., Yang, T. H., Messing, R. O., Ching, Y. H., Lin, S. C., and Chern, Y. (1997) J. Biol. Chem. 272, 4970-4977[Abstract/Free Full Text]
  40. Kiang, J. G., Gist, I. D., and Tsokos, G. C. (2000) Mol. Cell. Biochem. 204, 169-178[CrossRef][Medline] [Order article via Infotrieve]
  41. Kiang, J. G., Kiang, S. C., Juang, Y. T., and Tsokos, G. C. (2002) Am. J. Physiol. 282, G415-G423
  42. Tanaka, M., Machida, Y., Niu, S., Ikeda, T., Jana, N. R., Doi, H., Kurosawa, M., Nekooki, M., and Nukina, N. (2004) Nat. Med. 10, 148-154[CrossRef][Medline] [Order article via Infotrieve]
  43. Chern, Y., Lai, H. L., Fong, J. C., and Liang, Y. (1993) Mol. Pharmacol. 44, 950-958[Abstract]
  44. Sugars, K. L., Brown, R., Cook, L. J., Swartz, J., and Rubinsztein, D. C. (2004) J. Biol. Chem. 279, 4988-4999[Abstract/Free Full Text]
  45. Ionescu, A. M., Schwarz, E. M., Vinson, C., Puzas, J. E., Rosier, R., Reynolds, P. R., and O'Keefe, R. J. (2001) J. Biol. Chem. 276, 11639-11647[Abstract/Free Full Text]
  46. Ionescu, A. M., Drissi, H., Schwarz, E. M., Kato, M., Puzas, J. E., McCance, D. J., Rosier, R. N., Zuscik, M. J., and O'Keefe, R. J. (2004) J. Cell. Physiol. 198, 428-440[CrossRef][Medline] [Order article via Infotrieve]
  47. Schaffar, G., Breuer, P., Boteva, R., Behrends, C., Tzvetkov, N., Strippel, N., Sakahira, H., Siegers, K., Hayer-Hartl, M., and Hartl, F. U. (2004) Mol. Cell 15, 95-105[CrossRef][Medline] [Order article via Infotrieve]
  48. Jiang, H., Nucifora, F. C., Jr., Ross, C. A., and DeFranco, D. B. (2003) Hum. Mol. Genet. 12, 1-12[Abstract/Free Full Text]
  49. Mantamadiotis, T., Lemberger, T., Bleckmann, S. C., Kern, H., Kretz, O., Martin Villalba, A., Tronche, F., Kellendonk, C., Gau, D., Kapfhammer, J., Otto, C., Schmid, W., and Schutz, G. (2002) Nat. Genet. 31, 47-54[CrossRef][Medline] [Order article via Infotrieve]
  50. Fitzpatrick, S. L., and Richards, J. S. (1993) Mol. Endocrinol. 7, 341-354[Abstract/Free Full Text]
  51. Wilson, T. E., Fahrner, T. J., and Milbrandt, J. (1993) Mol. Cell. Biol. 13, 5794-5804[Abstract/Free Full Text]
  52. Clemens, J. W., Lala, D. S., Parker, K. L., and Richards, J. S. (1994) Endocrinology 134, 1499-1508[Abstract/Free Full Text]
  53. Chen, J. F., Xu, K., Petzer, J. P., Staal, R., Xu, Y. H., Beilstein, M., Sonsalla, P. K., Castagnoli, K., Castagnoli, N., Jr., and Schwarzschild, M. A. (2001) J. Neurosci. 21, 1-6[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Neurosci.Home page
S.-Y. Chou, J.-Y. Weng, H.-L. Lai, F. Liao, S. H. Sun, P.-H. Tu, D. W. Dickson, and Y. Chern
Expanded-Polyglutamine Huntingtin Protein Suppresses the Secretion and Production of a Chemokine (CCL5/RANTES) by Astrocytes
J. Neurosci., March 26, 2008; 28(13): 3277 - 3290.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. ProteomicsHome page
M.-C. Chiang, C.-G. Juo, H.-H. Chang, H.-M. Chen, E. C. Yi, and Y. Chern
Systematic Uncovering of Multiple Pathways Underlying the Pathology of Huntington Disease by an Acid-cleavable Isotope-coded Affinity Tag Approach
Mol. Cell. Proteomics, May 1, 2007; 6(5): 781 - 797.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
M.-C. Chiang, H.-M. Chen, Y.-H. Lee, H.-H. Chang, Y.-C. Wu, B.-W. Soong, C.-M. Chen, Y.-R. Wu, C.-S. Liu, D.-M. Niu, et al.
Dysregulation of C/EBP{alpha} by mutant Huntingtin causes the urea cycle deficiency in Huntington's disease
Hum. Mol. Genet., March 1, 2007; 16(5): 483 - 498.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
280/14/14331    most recent
M413279200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chiang, M.-C.
Right arrow Articles by Chern, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chiang, M.-C.
Right arrow Articles by Chern, Y.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2005 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement