Combinatorial Transcription Factor Regulation of the Cyclic AMP-response Element on the Pgc-1α Promoter in White 3T3-L1 and Brown HIB-1B Preadipocytes
2009; Elsevier BV; Volume: 284; Issue: 31 Linguagem: Inglês
10.1074/jbc.m109.021766
ISSN1083-351X
AutoresAngeliki Karamitri, Andrew Shore, Kevin Docherty, John R. Speakman, Michael A. Lomax,
Tópico(s)Cardiovascular Disease and Adiposity
ResumoCold stress in rodents increases the expression of UCP1 and PGC-1α in brown and white adipose tissue. We have previously reported that C/EBPβ specifically binds to the CRE on the proximal Pgc-1α promoter and increases forskolin-sensitive Pgc-1α and Ucp1 expression in white 3T3-L1 preadipocytes. Here we show that in mice exposed to a cold environment for 24 h, Pgc-1α, Ucp1, and C/ebpβ but not C/ebpα or C/ebpδ expression were increased in BAT. Conversely, expression of the C/EBP dominant negative Chop10 was increased in WAT but not BAT during cold exposure. Reacclimatization of cold-exposed mice to a warm environment for 24 h completely reversed these changes in gene expression. In HIB-1B, brown preadipocytes, forskolin increased expression of Pgc-1α, Ucp1, and C/ebpβ early in differentiation and inhibited Chop10 expression. Employing chromatin immunoprecipitation, we demonstrate that C/EBPβ, CREB, ATF-2, and CHOP10 are bound to the Pgc-1α proximal CRE, but CHOP10 does not bind in HIB-1B cell lysates. Forskolin stimulation and C/EBPβ overexpression in 3T3-L1 cells increased C/EBPβ and CREB but displaced ATF-2 and CHOP10 binding to the Pgc-1α proximal CRE. Overexpression of ATF-2 and CHOP10 in 3T3-L1 cells decreased Pgc-1α transcription. Knockdown of Chop10 in 3T3-L1 cells using siRNA increased Pgc-1α transcription, whereas siRNA against C/ebpβ in HIB-1B cells decreased Pgc-1α and Ucp1 expression. We conclude that the increased cAMP stimulation of Pgc-1α expression is regulated by the combinatorial effect of transcription factors acting at the CRE on the proximal Pgc-1α promoter. Cold stress in rodents increases the expression of UCP1 and PGC-1α in brown and white adipose tissue. We have previously reported that C/EBPβ specifically binds to the CRE on the proximal Pgc-1α promoter and increases forskolin-sensitive Pgc-1α and Ucp1 expression in white 3T3-L1 preadipocytes. Here we show that in mice exposed to a cold environment for 24 h, Pgc-1α, Ucp1, and C/ebpβ but not C/ebpα or C/ebpδ expression were increased in BAT. Conversely, expression of the C/EBP dominant negative Chop10 was increased in WAT but not BAT during cold exposure. Reacclimatization of cold-exposed mice to a warm environment for 24 h completely reversed these changes in gene expression. In HIB-1B, brown preadipocytes, forskolin increased expression of Pgc-1α, Ucp1, and C/ebpβ early in differentiation and inhibited Chop10 expression. Employing chromatin immunoprecipitation, we demonstrate that C/EBPβ, CREB, ATF-2, and CHOP10 are bound to the Pgc-1α proximal CRE, but CHOP10 does not bind in HIB-1B cell lysates. Forskolin stimulation and C/EBPβ overexpression in 3T3-L1 cells increased C/EBPβ and CREB but displaced ATF-2 and CHOP10 binding to the Pgc-1α proximal CRE. Overexpression of ATF-2 and CHOP10 in 3T3-L1 cells decreased Pgc-1α transcription. Knockdown of Chop10 in 3T3-L1 cells using siRNA increased Pgc-1α transcription, whereas siRNA against C/ebpβ in HIB-1B cells decreased Pgc-1α and Ucp1 expression. We conclude that the increased cAMP stimulation of Pgc-1α expression is regulated by the combinatorial effect of transcription factors acting at the CRE on the proximal Pgc-1α promoter. A remarkable feature of rodent adipose tissue is the ability to undergo a transition from white to brown adipose during cold exposure. This transition is driven by the sympathetic nervous system and enables the animal to defend its body temperature by increasing heat production due to the oxidation of fatty acids by brown adipose mitochondria, which contain the tissue-specific UCP1 (uncoupling protein 1). The inherent plasticity that allows this transition from white to brown adipose tissue is not due to a change in the number of adipocytes in a depot but to modification of white adipocytes to the brown adipocyte phenotype, sometimes referred to as “transdifferentiation” (1Cinti S. Nutr. Metab. Cardiovasc Dis. 2006; 16: 569-574Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). This change in adipocyte cell type is orchestrated by the sympathetic nervous system neurotransmitter, noradrenaline, which stimulates intracellular cAMP and activates a protein kinase A-dependent increase in UCP1 expression. Transdifferentiation from white to brown adipose tissue and/or vice versa has been reported in many species, including rodents, cats, dogs, ruminants (2Loncar D. Cell Tissue Res. 1991; 266: 149-161Crossref PubMed Scopus (150) Google Scholar, 3Ashwell M. Stirling D. Freeman S. Holloway B.R. Int. J. Obes. 1987; 11: 357-365PubMed Google Scholar, 4Casteilla L. Champigny O. 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The proposal that BAT is present in adult humans (1Cinti S. Nutr. Metab. Cardiovasc Dis. 2006; 16: 569-574Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) has recently been supported by the use of fluorodeoxyglucose positron emission tomography to reveal BAT human depots localized to the supraclavicular, neck, spinal cord, and mediastinum areas (14Nedergaard J. Bengtsson T. Cannon B. Am. J. Physiol. Endocrinol. 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Within 6 h of adding the hormonal mixture to induce differentiation, there is an increased expression of C/EBPβ, but this lacks DNA binding activity until much later due to the simultaneous expression of CHOP10 (C/EBP homologous protein), which sequesters and inactivates C/EBPβ by heterodimerization with its leucine zipper (31Tang Q.Q. Lane M.D. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 12446-12450Crossref PubMed Scopus (142) Google Scholar). After a lag period, CHOP10 undergoes down-regulation, releasing C/EBPβ from inhibitory constraint, allowing transactivation of the C/EBPα and PPARγ genes, transcription factors required for terminal differentiation. The transcriptional events involved in brown adipocyte differentiation are not as well characterized, but genetic ablation studies have demonstrated that PPARγ, C/EBPβ, and C/EBPδ but not C/EBPα are also essential for this process (32Nedergaard J. Petrovic N. Lindgren E.M. Jacobsson A. Cannon B. Biochim. Biophys. 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Here we provide evidence that conversion of WAT to BAT during exposure to a cold environment involves combinatorial transcriptional factor regulation of the Pgc-1α proximal CRE, which gives rise to adipose tissue-specific expression of Pgc-1α and Ucp1 during stimulation by cAMP. Three groups of C57BL/6 mice were used, each consisting of four female individuals. All three groups were housed individually in cages measuring 48 × 15 × 13 cm with a 16-h light and 8-h dark cycle with access to bedding material. All groups had access ad libitum to a standard mouse chow diet. Mouse weight and feed consumption was measured at 24-h intervals. One group was kept at 22 ± 2 °C for 72 h; these mice comprised the warm acclimatized group. A second group was kept at 22 ± 2 °C for 48 h, followed by 8 ± 2 °C for 24 h and comprised the cold acclimatized group. The third group of 12 individuals was kept at 22 ± 2 °C for 24 h and then at 8 ± 2 °C for 24 h, followed by a return to 22 ± 2 °C for 24 h and comprised the reacclimatized group. All experiments followed institutional guidelines at the University of Aberdeen as well as those set out for animal care by the United Kingdom Home Office. Animals were euthanized by concussion, followed by cervical dislocation following Home Office guidelines. The firefly luciferase reporter gene construct containing 264 bp (264PGC1α-pGL3) from the region upstream of the rat Pgc-1α transcription start site ligated to the pGL3-Basic vector (Promega) has been described (25Karamanlidis G. Karamitri A. Docherty K. Hazlerigg D.G. Lomax M.A. J. Biol. Chem. 2007; 282: 24660-24669Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). The pMSVC/EBPβ (mouse) expression plasmid that contains the respective cDNAs under the control of the mouse sarcoma virus long terminal repeat was kindly provided by S. McKnight (University of Texas Southwestern Medical Center, Dallas, TX). The mock plasmid pcDNA3 was from Invitrogen. The CRE-positive vector (pCRE-LUC) that contains four juxtapose copies of the consensus CRE sequence upstream of a TATA box to drive expression of the firefly luciferase gene was purchased from Stratagene. Expression vector for the truncated isoform (pSG/LIP) of C/EBPβ was provided by Birgit Gellersen (Institute for Hormone and Fertility Research, University of Hamburg, Hamburg, Germany). The expression plasmids pEBG2T-ATF2, which contained human ATF-2, and pCMV5CREB1, which contained human CREB1, were kindly provided by Philip Cohen (University of Dundee, UK). Mouse CHOP10 wild type and dominant negative (deletion of the leucine zipper domain) plasmids were supplied by David Ron (New York, NY). 3T3-L1 cells (ECACC) and HIB-1B cells (kindly provided by B. Spiegelman) were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum (Invitrogen) in 5% CO2. For differentiation, HIB-1B cells were cultured to confluence (day 0), and then they were exposed to the differentiation mixture (0.5 mm isobutylmethylxanthine, 250 nm dexamethasone, 170 nm insulin, 10 nm T3). After 48 h, cells were maintained in medium containing 5% fetal bovine serum, 170 nm insulin, and 10 nm T3 until day 7 for harvest, and this medium was replaced daily. HIB-1B cells were transfected with luciferase plasmids using Fugene 6 (Roche Applied Science) at a charge ratio of 3:1, and 3T3-L1 cells were transfected with Lipofectamine 2000 (Invitrogen) at a charge ratio of 2:1 at 80% confluence in combination with expression vectors, where indicated, or pcDNA3 as a control. The pRL-SV40 (from Promega) that carries Renilla luciferase was also co-transfected as an internal control for monitoring the transfection efficiency. Thirty-six hours later, cells were treated with forskolin in serum-free conditions, and after 12 h, cells were harvested, and luciferase activities were analyzed using the Dual-Luciferase assay kit (Promega), as recommended by the manufacturer. Values were expressed relative to the control Renilla to allow for differences in transfection efficiency. HIB-1B and 3T3-L1 preadipocytes were grown in CultureWell™ chambered coverglasses (Invitrogen). Cells were treated with 10 μm forskolin or vehicle solution (DMSO) for 30 min and fixed with 4% paraformaldehyde in phosphate-buffered saline for 15 min. Cells were permeabilized with 0.3% (v/v) Triton X-100 for 5 min and blocked with 3% (w/v) bovine serum albumin for 1 h. After blocking, cells were incubated in 3% bovine serum albumin in phosphate-buffered saline containing 0.1% Tween 20 and anti-pCREB, anti-pATF-2 (Cell Signaling), or anti-C/EBPβ (C-19; Santa Cruz Biotechnology) overnight at 4 °C. Cells were washed three times with 0.1% Tween 20 and incubated at room temperature for 1 h in the dark after adding fluorescence-labeled secondary antibody (fluorescein isothiocyanate-conjugated anti-rabbit IgG from Sigma). After washing in phosphate-buffered saline, antifade solution with 4′,6-diamidino-2-phenylindole (Molecular Probes) was applied, and cells were covered with mounting solution until they were examined by fluorescence microscopy. Commercially available mouse siRNA oligonucleotides targeting C/EBPβ and CHOP10 as well as control (non-targeting) siRNA (Dharmacon) were employed to transfect HIB-1B and 3T3-L1 cells (100 nm siRNA), respectively, by the use of Dharmafect 3 (Dharmacon) according to the manufacturer's protocol. In cotransfection experiments involving siRNAs, the siRNA was added to the well when cells reached 60% confluence, and plasmid transfection was performed 24 h later. HIB-1B cells were kept in serum-free medium for 4 h after transfection, which was replaced with medium containing 20% fetal bovine serum to maximize cell growth and prevent potential cytotoxicity. Cells were harvested 72 h after transfection for RNA extraction. Chromatin immunoprecipitation (ChIP) assays were performed according to the manufacturer's protocol (Upstate Biotechnology, Inc., Lake Placid, NY). Briefly, HIB-1B and 3T3-L1 preadipocytes were transfected with pcDNA3 or pMSVC/EBPβ, and 48 h later (at confluence), they were stimulated with forskolin (10 μm) or DMSO for 1 h. Protein-DNA cross-linking was achieved by adding formaldehyde (1% final concentration) for 1 h at 37 °C. The cells were washed twice with ice-cold phosphate-buffered saline and lysed in SDS lysis buffer (3% SDS, 1% Triton X-100, 0.5% sodium deoxycholate, 10 mm EDTA, and 50 mm Tris-HCl, pH 8.1) supplemented with protease inhibitors. The whole cell lysates were sonicated with a Soniprep 150 for 30 s at the maximum setting. This was repeated eight times with 1-min intervals between each 30-s pulse, yielding chromatin fragments between 200 and 500 bp in size. Lysates were centrifuged at 13,000 rpm for 10 min, and the resulting supernatants were diluted 10-fold with ChIP dilution buffer in the presence of protease inhibitors. Normal rabbit IgG (sc-2027; Santa Cruz Biotechnology) and salmon sperm DNA, 50% protein A-agarose slurry (80 μl) was added to the lysates, which were incubated for 30 min at 4 °C to reduce nonspecific background. Agarose beads were precipitated by brief centrifugation, and the supernatant was collected. Antibodies against CREB (Upstate Biotechnology), C/EBPβ, ATF-2 (N-96; Santa Cruz Biotechnology), CHOP10 (F-168; Santa Cruz Biotechnology), or preimmune serum (negative control) were added to the 2-ml supernatant fraction, and the mixture was incubated overnight at 4 °C with rotation. The immunocomplexes were collected by binding to 60 μl of salmon sperm DNA-Protein A-agarose slurry after incubation for 1 h at 4 °C with rotation. The antibody-histone-DNA complexes were washed sequentially with low salt buffer, high salt buffer, lithium chloride wash buffer, and TE buffer. The pellets were then eluted, and reversal of cross-linking was done by heating at 65 °C overnight in the presence of NaCl. Purification of DNA was done with a QIAquick PCR purification kit (Qiagen), and the obtained DNA fragments were analyzed by PCR using the following primer pairs for the mouse Pgc-1α promoter: forward, 5′-GGGCTGCCTTGGAGTGACGTC-3′; reverse, 5′-AGTCCCCAGTCACATGACAAAG-3′. Western blotting on whole cell lysates was performed as reported previously (25Karamanlidis G. Karamitri A. Docherty K. Hazlerigg D.G. Lomax M.A. J. Biol. Chem. 2007; 282: 24660-24669Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Immunological detection was performed using antibodies directed against CREB (1:1000 dilution), ATF-2 (1:500 dilution), CHOP10 (1:350 dilution), C/EBPβ (1:500 dilution), and actin (1:250 dilution; Sigma). The antigen-antibody complex was detected by incubating the membrane for 1 h at room temperature in buffer containing a 1:1000 dilution of horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (Cell Signaling) and visualized with SuperSignal West Pico blotting substrate (Pierce). Total RNA was extracted from cultured cells by use of TRI reagent (Sigma). Prior to RT-PCR, samples were treated with DNA-free DNase to remove contaminating genomic or plasmid DNA. cDNA was generated using the cDNA synthesis kit from Qiagen. Quantitative real time PCR (qRT-PCR) was performed using SYBR Green (Qiagen) according to the manufacturer's instructions in a Rotor Gene 3000 thermal cycler (Corbert Research). The sequences of the primers used for real time PCR were Pgc-1α sense (GCGCCGTGTGATTTACGTT) and antisense (AAAACTTCAAAGCGGTCTCTCAA), Ucp1 sense (CCTGCCTCTCTCGGAAACAA) and antisense (TGTAGGCTGCCCAATGAACA), C/ebpα sense (CCGGGAGAACTCTAACTC) and antisense (GATGTAGGCGCTGATGT), C/ebpβ sense (GCAAGAGCCGCGACAAG) and antisense (GGCTCGGGCAGCTGCTT), C/ebpδ sense (ACGACGAGAGCGCCATC) and antisense (TCGCCGTCGCCCCAGTC), Chop10 sense (AGAGGAAGAATCAAAAACCTTCACT) and antisense (ACTCTGTTTCCGTTTCCTAGTTCTT), and 18 S rRNA sense (GTAACCCGTTGAACCCCATT) and antisense (CCATCCAATCGGTAGTAGCG). Expression levels for all genes were normalized to the internal control 18 S rRNA. All of the data were analyzed with either Student's t test or two-way analysis of variance. We have previously demonstrated that C/EBPβ overexpression was able to increase Pgc-1α and Ucp1 expression in response to cAMP stimulation in 3T3-L1 cells, suggesting that C/EBPβ is implicated in sympathetic neural stimulation of brown adipose tissue recruitment in white adipose tissue depots. We first set out to demonstrate that the increase in Pgc-1α and Ucp1 expression in adipose tissue in response to cold stress was associated with increased C/ebpβ expression. Maintaining mice for 24 h in a cold environment at 8 °C compared with 22 °C significantly increased mRNA levels for both Pgc-1α (p < 0.05) and Ucp1 (p < 0.01; Fig. 1, A and B) in interscapular BAT (iBAT), and these changes were reversed within 24 h of reacclimatization of cold-maintained animals back to a warm environment. Interscapular WAT (iWAT) represents tissue that was distinguishable from iBAT by visual dissection, and as expected, both Pgc-1α and Ucp1 expression were lower compared with iBAT. The increase in Pgc-1α and Ucp1 expression in iWAT (p < 0.01) in animals maintained in the cold was presumably due to a population of iWAT contaminated with iBAT (Fig. 1, A and B). There was a small but significant (p < 0.01) induction of Ucp1 expression in gonadal WAT, which is likely to be due to BAT recruitment of small pockets of cells previously observed in WAT (Fig. 1A). Ucp1 was not expressed in liver. Pgc-1α was expressed at lower levels in gonadal WAT and liver and was not altered by housing temperature. When tissue mRNA for the three C/EBP isomers was measured (Fig. 1, C–E), there were no significant effects of housing temperature on C/ebpα and C/ebpδ levels in any of the tissues studied, but C/ebpβ expression was significantly (p < 0.01) increased by the cold housing treatment in iBAT, iWAT, and liver but not gonadal WAT. Similar to Pgc-1α and Ucp1, levels of C/ebpβ mRNA were highest in iBAT, and responses to the cold environment were rapidly reversed by 24-h reacclimatization in the warmth. Exposure to cold has previously been reported to increase C/ebpβ but not C/ebpα or C/ebpδ in iBAT (37Rehnmark S. Antonson P. Xanthopoulos K.G. Jacobsson A. FEBS Lett. 1993; 318: 235-241Crossref PubMed Scopus (36) Google Scholar, 38Manchado C. Yubero P. Viñas O. Iglesias R. Villarroya F. Mampel T. Giralt M. Biochem. J. 1994; 302: 695-700Crossref PubMed Scopus (36) Google Scholar), indicating a positive association of C/EBPβ gene expression with Pgc-1α and Ucp1. We next assessed the levels of Chop10, which has been reported to act as a dominant negative transcription factor of C/EBPs (Fig. 1F); Chop10 was not altered by environmental temperature in liver and decreased in iBAT (p = 0.05) but was increased (p < 0.01) by the cold treatment in gonadal WAT and iWAT. Having demonstrated that changes in adipose tissue expression of C/ebpβ in response to transitions from warm to cold and cold to warm environments followed the same pattern as Pgc-1α and Ucp1 expression, we next sought to examine these associations during differentiation in the brown adipocyte cell line, HIB-1B. Expression of Ucp1 was significantly (p < 0.01) increased by treatment with forskolin, compared with basal DMSO-treated cells at confluence (Fig. 2A), as we have previously demonstrated (25Karamanlidis G. Karamitri A. Docherty K. Hazlerigg D.G. Lomax M.A. J. Biol. Chem. 2007; 282: 24660-24669Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). This response disappeared 12 and 24 h after inducing differentiation but returned by 36 h and increased to 7 days after induction, when lipid droplets can be observed in HIB-1B cells. Basal Ucp1 mRNA expression was low throughout the differentiation protocol unless stimulated by cAMP with forskolin (39Ross S.R. Choy L. Graves R.A. Fox N. Solevjeva V. Klaus S. Ricquier D. Spiegelman B.M. Proc. Natl. Acad. Sci. U.S.A. 1992; 89: 7561-7565Crossref PubMed Scopus (113) Google Scholar). Forskolin-induced expression of Pgc-1α followed a pattern similar to that of Ucp1 (Fig. 2B), with a significant increase (p < 0.01) at confluence followed by a loss of response to forskolin at 12 h pos
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