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• W2103102045 abstract "Cells use redox signaling to adapt to oxidative stress. For instance, certain transcription factors exist in a latent state that may be disrupted by oxidative modifications that activate their transcription potential. We hypothesized that DNA-binding sites (response elements) for redox-sensitive transcription factors may also exist in a latent state, maintained by co-repressor complexes containing class I histone deacetylase (HDAC) enzymes, and that HDAC inactivation by oxidative stress may antagonize deacetylase activity and unmask electrophile-response elements, thus activating transcription. Electrophiles suitable to test this hypothesis include reactive carbonyl species, often derived from peroxidation of arachidonic acid. We report that α,β-unsaturated carbonyl compounds, e.g. the cyclopentenone prostaglandin, 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2), and 4-hydroxy-2-nonenal (4HNE), alkylate (carbonylate), a subset of class I HDACs including HDAC1, -2, and -3, but not HDAC8. Covalent modification at two conserved cysteine residues, corresponding to Cys261 and Cys273 in HDAC1, coincided with attenuation of histone deacetylase activity, changes in histone H3 and H4 acetylation patterns, derepression of a LEF1·β-catenin model system, and transcription of HDAC-repressed genes, e.g. heme oxygenase-1 (HO-1), Gadd45, and HSP70. Identification of particular class I HDACs as components of the redox/electrophile-responsive proteome offers a basis for understanding how cells stratify their responses to varying degrees of pathophysiological oxidative stress associated with inflammation, cancer, and metabolic syndrome. Cells use redox signaling to adapt to oxidative stress. For instance, certain transcription factors exist in a latent state that may be disrupted by oxidative modifications that activate their transcription potential. We hypothesized that DNA-binding sites (response elements) for redox-sensitive transcription factors may also exist in a latent state, maintained by co-repressor complexes containing class I histone deacetylase (HDAC) enzymes, and that HDAC inactivation by oxidative stress may antagonize deacetylase activity and unmask electrophile-response elements, thus activating transcription. Electrophiles suitable to test this hypothesis include reactive carbonyl species, often derived from peroxidation of arachidonic acid. We report that α,β-unsaturated carbonyl compounds, e.g. the cyclopentenone prostaglandin, 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2), and 4-hydroxy-2-nonenal (4HNE), alkylate (carbonylate), a subset of class I HDACs including HDAC1, -2, and -3, but not HDAC8. Covalent modification at two conserved cysteine residues, corresponding to Cys261 and Cys273 in HDAC1, coincided with attenuation of histone deacetylase activity, changes in histone H3 and H4 acetylation patterns, derepression of a LEF1·β-catenin model system, and transcription of HDAC-repressed genes, e.g. heme oxygenase-1 (HO-1), Gadd45, and HSP70. Identification of particular class I HDACs as components of the redox/electrophile-responsive proteome offers a basis for understanding how cells stratify their responses to varying degrees of pathophysiological oxidative stress associated with inflammation, cancer, and metabolic syndrome. Cellular oxidative stress can vary widely in severity and scope. Consequently, redox signaling must accommodate physiological demands from respiration, metabolism, host defense, cell replication, and aging plus demands from pathological oxidative stress encountered during inflammation, malignancy, reperfusion injury, and metabolic syndrome. Stimulus-response coupling in these different situations must be properly stratified; otherwise, maladaptation can have grave outcomes. An insufficient response to oxidative stress can lead to cell death, which typifies many neurodegenerative diseases. An excessive response to oxidative stress can lead to hypertrophy, hyperplasia, or neoplasia (1Medzhitov R. Nature. 2008; 454: 428-435Crossref PubMed Scopus (3617) Google Scholar). Phenotypic adaptation to oxidative stress derives, in part, from the expression of genes to protect cells from damage, to repair damage, and to bolster their survival. This involves cellular proteins collectively termed the redox/electrophile-responsive proteome (2Stamatakis K. Pérez-Sala D. Ann. N.Y. Acad. Sci. 2006; 1091: 548-570Crossref PubMed Scopus (44) Google Scholar, 3Oh J.Y. Giles N. Landar A. Darley-Usmar V. Biochem. J. 2008; 411: 297-306Crossref PubMed Scopus (100) Google Scholar). These proteins vary widely in cellular localization and functionality, but all have cysteine residues with distinctively nucleophilic thiols (pKa ≤ 5), which are readily oxidized to sulfenic/sulfinic acids by reactive oxygen species (ROS) 2The abbreviations used are: ROSreactive oxygen speciesRCSreactive carbonyl speciesPGprostaglandin15d-PGJ215-deoxy-Δ12,14-PGJ215d-PGJ2-B15-deoxy-Δ12,14-PGJ2-biotinHDAChistone deacetylase4HNE4-hydroxy-2-nonenalSTFSuperTOPflash®TSAtrichostatin AWTwild typeFCSfetal calf serumPBSphosphate-buffered salineLEFlymphoid enhancer factorTCFT-cell factor. or readily alkylated by reactive carbonyl species (RCS) (4Suzuki M. Mori M. Niwa T. Hirata R. Furuta K. Ishikawa T. Noyori R. J. Am. Chem. Soc. 1997; 119: 2376-2385Crossref Scopus (131) Google Scholar, 5Salsbury Jr., F.R. Knutson S.T. Poole L.B. Fetrow J.S. Protein Sci. 2008; 17: 299-312Crossref PubMed Scopus (103) Google Scholar). RCS originate from either non-enzymatic or enzymatic peroxidation of lipids (especially arachidonic acid), which generates α,β-unsaturated aldehydes (enals) (e.g. 4-hydroxy-2-nonenal (4HNE), crotonaldehyde, acrolein) and α,β-unsaturated ketones (enone) (e.g. cyclopentenone prostaglandins). Post-translational covalent modification of cysteinyl thiols by RCS has been termed “carbonylation” (6Grimsrud P.A. Xie H. Griffin T.J. Bernlohr D.A. J. Biol. Chem. 2008; 283: 21837-21841Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar), and carbonylation of proteins is a distinctive feature of cellular redox signaling by peroxiredoxins (7Poole L.B. Karplus P.A. Claiborne A. Annu. Rev. Pharmacol. Toxicol. 2004; 44: 325-347Crossref PubMed Scopus (489) Google Scholar, 8Cordray P. Doyle K. Edes K. Moos P.J. Fitzpatrick F.A. J. Biol. Chem. 2007; 282: 32623-32629Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), tyrosine phosphatases (9Salmeen A. Andersen J.N. Myers M.P. Meng T.C. Hinks J.A. Tonks N.K. Barford D. Nature. 2003; 423: 769-773Crossref PubMed Scopus (761) Google Scholar, 10Lu C. Chan S.L. Fu W. Mattson M.P. J. Biol. Chem. 2002; 277: 24368-24375Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), and kinases (11Wagner T.M. Mullally J.E. Fitzpatrick F.A. J. Biol. Chem. 2006; 281: 2598-2604Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 12Rossi A. Kapahi P. Natoli G. Takahashi T. Chen Y. Karin M. Santoro M.G. Nature. 2000; 403: 103-108Crossref PubMed Scopus (1198) Google Scholar) and transcription factors (p53, NFκB, Nrf2). reactive oxygen species reactive carbonyl species prostaglandin 15-deoxy-Δ12,14-PGJ2 15-deoxy-Δ12,14-PGJ2-biotin histone deacetylase 4-hydroxy-2-nonenal SuperTOPflash® trichostatin A wild type fetal calf serum phosphate-buffered saline lymphoid enhancer factor T-cell factor. Overall, we have a rudimentary understanding of how ROS and RCS integrate the actions of membrane and cytosolic proteins to govern redox-responsive transcription. By contrast, we know very little about the actions of RCS on nuclear proteins and processes beyond the investigations of Narumiya et al. (13Narumiya S. Ohno K. Fukushima M. Fujiwara M. J. Pharmacol. Exp. Ther. 1987; 242: 306-311PubMed Google Scholar, 14Narumiya S. Ohno K. Fujiwara M. Fukushima M. J. Pharmacol. Exp. Ther. 1986; 239: 506-511PubMed Google Scholar, 15Narumiya S. Fukushima M. J. Pharmacol. Exp. Ther. 1986; 239: 500-505PubMed Google Scholar), who first reported that cyclopentenone prostaglandins concentrated within the nucleus of cells via irreversible binding to unidentified nuclear and chromatin-associated proteins. The findings by Narumiya et al. (13Narumiya S. Ohno K. Fukushima M. Fujiwara M. J. Pharmacol. Exp. Ther. 1987; 242: 306-311PubMed Google Scholar, 14Narumiya S. Ohno K. Fujiwara M. Fukushima M. J. Pharmacol. Exp. Ther. 1986; 239: 506-511PubMed Google Scholar, 15Narumiya S. Fukushima M. J. Pharmacol. Exp. Ther. 1986; 239: 500-505PubMed Google Scholar) and the biological importance of proper stimulus-response coupling in cells exposed to oxidative stress prompted our hypothesis that regulation of redox-responsive transcription factors might coincide with redox regulation of proteins that alter chromatin dynamics. Prominent among these are histone deacetylases, which limit access to response elements on DNA within heterochromatin, thereby repressing gene expression. Mammalian class I HDAC1, -2, -3, and -8 are homologous to yeast RPD3 (16Taunton J. Hassig C.A. Schreiber S.L. Science. 1996; 272: 408-411Crossref PubMed Scopus (1514) Google Scholar), reside in the nucleus, and may be incorporated into multiprotein gene repression complexes and co-repressor complexes such as mSin3a (17Hassig C.A. Schreiber S.L. Curr. Opin. Chem. Biol. 1997; 1: 300-308Crossref PubMed Scopus (335) Google Scholar, 18Hassig C.A. Fleischer T.C. Billin A.N. Schreiber S.L. Ayer D.E. Cell. 1997; 89: 341-347Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar), NuRD (19Knoepfler P.S. Eisenman R.N. Cell. 1999; 99: 447-450Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar), and N-CoR/SMRT (20Struhl K. Genes Dev. 1998; 12: 599-606Crossref PubMed Scopus (1531) Google Scholar, 21Jepsen K. Rosenfeld M.G. J. Cell Sci. 2002; 115: 689-698Crossref PubMed Google Scholar). Repression and derepression of the lymphoid enhancer factor/T-cell factor (LEF/TCF) family of transcriptional activators/inhibitors by the recruitment of class I histone deacetylases (HDACs) typify this process (22Courey A.J. Jia S. Genes Dev. 2001; 15: 2786-2796PubMed Google Scholar, 23Billin A.N. Thirlwell H. Ayer D.E. Mol. Cell. Biol. 2000; 20: 6882-6890Crossref PubMed Scopus (189) Google Scholar). We report that RCS covalently modified a subset of class I HDACs including HDAC1, -2, and -3 as well as the yeast homologue RPD3, but not HDAC8. Covalent modification of two conserved cysteines, Cys261 and Cys273 in HDAC1, occurred in a dose- and time-dependent manner that led to attenuation of histone deacetylase, changes in histone H3 and H4 acetylation patterns, derepression of LEF1·β-catenin transactivation, and activation of RCS-sensitive genes repressed by HDACs. Our results imply that HDAC1, -2, and -3 isoenzymes are components of a redox/electrophile-responsive proteome that governs redox signaling in eukaryotes. Our data also suggest a mechanism by which cells might stratify their responses and adapt to varying intensities of oxidative stress and may explain, in part, how maladaptation can influence the etiology of cancer, atherosclerosis, or other chronic diseases. Cyclopentenone prostaglandins including biotinylated derivatives, 4HNE, and HDAC inhibitor trichostatin A (TSA) were from Cayman Chemical (Ann Arbor, MI). NeutrAvidin agarose resin was purchased from Thermo Fisher Scientific. Primary antibodies for HDAC1 (H3284), HDAC2 (H3159), HDAC8 (H8038), and FLAG-M2 (F3165) as well as the HDAC inhibitor sodium butyrate were all from Sigma. Anti-HDAC3 (ab47237) was from Abcam (Cambridge, MA). Anti-acetyl-histone H3K14 (06-911), anti-Myc tag (05-724), and anti-acetyl-histone H4 (06-598) were all from Millipore (Billerica, MA). Anti-acetyl-histone H3K9 (9671) was from Cell Signaling (Danvers, MA). Horseradish peroxidase-conjugated secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Polyvinylidene difluoride membranes and Western Lightning chemiluminescence reagents were from PerkinElmer Life Sciences. Lipofectamine 2000 was from Invitrogen. Mammalian cell lysis buffer for biotin capture by NeutrAvidin pulldown as well as for histone acetylation assays contained 50 mm Tris, pH 7.4, 0.1 m NaCl, 2 mm EDTA, 1 mm NaF, and 1× CompleteTM protease inhibitor (Roche Applied Science) and 1% Triton X-100. Yeast lysis buffer contains 12% glycerol, 50 mm Tris, pH 7.5, 500 mm NaCl, 0.1% Triton X-100, 0.5 mm EDTA, and 1× CompleteTM protease inhibitor (Roche Applied Science). Reporter lysis buffer and luciferase assay system were from Promega (Madison, WI). Protein concentration from cell lysates was measured via Bradford analysis using the Bio-Rad protein assay solution (500-0006). All mammalian cell lines were propagated in medium containing penicillin and streptomycin and 2 mml-glutamine. SuperTOPflash® (STF) and STF3a (HEK293) cell lines were maintained in advanced Dulbecco's minimum essential medium and 2% FCS (HyClone); A549 cells were grown in F-12 medium with 10% FCS; HL-60 cells were grown in RPMI with 10% FCS; and HCT116 cells were grown in McCoy's 5A medium with 10% FCS. Yeast cells were a gift from Dr. David Stillman, University of Utah (strain DY6092), grown in YP medium containing 2% glucose and at 30 °C. Adherent mammalian cell lines were grown to ∼90% confluency in 35-mm plates containing 1–2% serum. Cells were treated with biotinylated prostaglandins for the times and concentrations indicated. Following treatment, cells were washed twice with PBS, pH 7.4, at 4 °C and harvested in 140 μl of lysis buffer. Cells are completely lysed with one freeze/thaw cycle, and cellular debris was pelleted at 10,000 × g. Between 100 and 200 μg of total protein was incubated overnight at 4 °C in 1 ml of total volume of PBS, pH 7.4, with 0.4% Tween 20 and 40 μl of NeutrAvidin bead slurry to sequester biotinylated (protein·prostaglandin-biotin) proteins. Following incubation, beads were pelleted and washed four times with PBS, pH 7.4, containing 0.4% Tween 20. Proteins were released from beads by boiling in 25 μl of 1.5× Laemmli buffer containing 3.75% β-mercaptoethanol for 5 min and assayed by Western immunoblot for individual HDACs. Histone acetylation was analyzed by Western blotting of 10 μg of total protein from whole cell lysates, separated on 10–20% SDS-PAGE gels. Saccharomyces cerevisiae cells harboring stably inserted RPD3-Myc (strain DY6092) were treated with 15d-PGJ2-B at A600 of ∼0.30–0.35 in 10 ml of medium at 30 °C for the time indicated. Yeast were pelleted and lysed using 0.7-mm zirconia beads (Biospec Products, Inc.) in lysis buffer. RPD3-Myc·15d-PGJ2-B conjugates were precipitated from lysates with NeutrAvidin beads as above and assayed by immunoblot for Myc tag. Total histone H4 acetylation was assayed by separating 15 μg of total protein from whole cell lysates on 10–20% SDS-PAGE gels followed by immunoblotting. MAD·LEF and MAD(Pro)·LEF chimeras, from Dr. Don Ayer, University of Utah School of Medicine, were prepared by cloning the first 105 bp of MAD upstream and in frame of full-length LEF1 (isoform 1) using EcoRI in expression vector pcDNA3.1. The MAD(Pro)·LEF mutant was prepared by mutating t35c (Leu12 to Pro12) and g46c (Ala16 to Pro16). We generated the Cys to Ser mutants of HDAC1-FLAG (from Eric Verdin, University of California San Francisco) via site-directed mutagenesis using a QuikChange site-directed mutagenesis kit (Stratagene) with the following forward primers and their reverse complement primers: C261S, 5′-CGGTGGTCTTACAGAGTGGCTCAGACTCC-3′; C273S, 5′-TATCTGGGGATCGGTTAGGTAGCTTCAATCTAACTATC-3′. We prepared 3H-acetylated histone substrate from RKO cells as described previously (24Sun J.M. Spencer V.A. Chen H.Y. Li L. Davie J.R. Methods. 2003; 31: 12-23Crossref PubMed Scopus (36) Google Scholar). Radio-labeled histones (∼500 dpm/μg) were purified by acid extraction and dialysis (25Shechter D. Dormann H.L. Allis C.D. Hake S.B. Nat. Protoc. 2007; 2: 1445-1457Crossref PubMed Scopus (682) Google Scholar) and lyophilized before use. Inhibition of HDAC activity was measured by treating recombinant HDAC3·N-CoR1 (5 ng/μl) in 60 μl of reaction buffer (50 mm Tris/Cl, pH 8.0, 137 mm NaCl, 2.7 mm KCl, 1 mm MgCl2, and 0.25 mg/ml BSA) with vehicle, HDAC inhibitors TSA, sodium butyrate, or 15d-PGJ2-B and 4HNE for 15 min followed by the addition of 3H-acetylated histone substrate (15,000 dpm/reaction dissolved in distilled H2O) and incubation at 37 °C for 30 min. The reaction was quenched with 10 μl of 10 n HCl/2.5 n acetic acid at 4 °C. Samples were extracted with 600 μl of ethyl acetate by vortexing for 10 s and centrifuged at 16,000 × g for 10 min. Released [3H]acetate was counted by adding 500 μl of the organic fraction to scintillation fluid. HDAC activity is measured as (counts (from test sample) (dpm) − counts (from vehicle control)(dpm))/((time (h)/volume of sample (ml)). STF cells grown to ∼60–70% confluency in 35-mm wells were treated with vehicle or 15d-PGJ2 (0–30 μm) in 2% FCS for 18 h. Cells were washed twice with PBS, pH 7.4, at 4 °C and lysed in 200 μl of reporter lysis buffer. Cell debris was pelleted at 10,000 × g, and 20 μg of total protein was used in a luciferase assay system (Promega). Vehicle signal was normalized to a value of 1 luciferase count per second. STF3a cells (Dr. David Virshup, Duke NUS Graduate Medical School) were used as described (26McCulloch M.W. Coombs G.S. Banerjee N. Bugni T.S. Cannon K.M. Harper M.K. Veltri C.A. Virshup D.M. Ireland C.M. Bioorg. Med. Chem. 2009; 17: 2189-2198Crossref PubMed Scopus (40) Google Scholar). STF3a cells (6 × 105 cells/well) were plated in 35-mm wells and grown overnight in antibiotic-free Dulbecco's minimum essential medium. MAD·LEF and MAD(Pro)·LEF chimeras were transfected (2 μg of DNA and 10 μl of Lipofectamine 2000) according to manufacturer's directions. After 24 h of incubation, cells were treated with vehicle, TSA (100 nm), butyrate (150 μm), 15d-PGJ2 (10 μm), Δ12-PGJ2 (10 μm), or 4HNE (10 μm) for 18 h. Cells were washed twice in PBS, pH 7.4, and lysed in 125 μl of reporter lysis buffer. Cell debris was pelleted at 10,000 × g, and 20 μg of total protein was used to measure luciferase activity (i.e. expression). Results, mean ± S.E. (n = 3–4) were normalized to a value of 100% in cells transfected with MAD(Pro)·LEF. A549 cells were grown to ∼70% confluency in 35-mm wells in antibiotic-free F-12 medium. HDAC1WT and HDAC1C261S/C273S plasmids were transfected (4 μg of DNA and 10 μl of Lipofectamine 2000), and medium was changed to 10% FCS after 6 h according to the manufacturer's directions. After 24 h, medium was changed to 1% FCS, and cells were treated with either vehicle or 15d-PGJ2 (10 μm). After 1 h, FCS was added to the cells to 10%, and the cells were incubated for 23 h before harvesting. Total RNA was isolated by using the RNeasy kit (Qiagen) according to manufacturer's directions followed by on-column DNase digestion. Total RNA was measured using a nanodrop spectrophotometer, and 2.5 μg of RNA was reverse-transcribed to cDNA using a first-strand cDNA synthesis kit (Fermentas) with oligo(dT)18 primers. The cDNA (1 μl) was used directly for quantitative PCR using SYBR Green master mix (Roche Applied Science) and the following primer pairs: HO-1, 5′-GTCTTCGCCCCTGTCTACTTC-3′, 5′-CTGGGCAATCTTTTTGAGCAC-3′; Gadd45, 5′-GAGAGCAGAAGACCGAAAGGA-3′, 5′-CACAACACCACGTTATCGGG-3′; HSP70, 5′-GCATCGAGACTATCGCTAATGAG-3′, 5′-TGCAAGGTTAGATTTTTCTGCCT-3′; GAPDH, 5′-ATGGGGAAGGTGAAGGTCG-3′, 5′-GGGGTCATTGATGGCAACAATA-3′; β-actin, 5′-TTCCTGGGCATGGAGTC-3′, 5′-CAGGTCTTTGCGGATGTC-3′. To investigate how RCS influence gene regulation via interaction with chromatin-associated proteins, we first tested whether HDACs are alkylated by cyclopentenone prostaglandins. PGD2 dehydrates spontaneously to form J-series enone and dienone prostaglandins (27Fitzpatrick F.A. Wynalda M.A. J. Biol. Chem. 1983; 258: 11713-11718Abstract Full Text PDF PubMed Google Scholar) (supplemental Fig. 1D), which can react with nucleophilic cysteinyl thiolates (28Noyori R. Suzuki M. Science. 1993; 259: 44-45Crossref PubMed Scopus (109) Google Scholar) found in transcription factors, e.g. NFκB (29Cernuda-Morollón E. Pineda-Molina E. Cañada F.J. Pérez-Sala D. J. Biol. Chem. 2001; 276: 35530-35536Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar), KEAP·NRF2 (3Oh J.Y. Giles N. Landar A. Darley-Usmar V. Biochem. J. 2008; 411: 297-306Crossref PubMed Scopus (100) Google Scholar), as well as unidentified chromatin-associated proteins (13Narumiya S. Ohno K. Fukushima M. Fujiwara M. J. Pharmacol. Exp. Ther. 1987; 242: 306-311PubMed Google Scholar). Hypothetically, any HDAC enzymes with redox-sensitive cysteinyl thiols should be prone to alkylation (carbonylation) by enones and enals. To test this hypothesis, we treated HEK293 cells with biotin analogs of 15d-PGJ2-B. The biotin epitope in PGJ congeners facilitates the isolation and identification of any cellular proteins they might alkylate (3Oh J.Y. Giles N. Landar A. Darley-Usmar V. Biochem. J. 2008; 411: 297-306Crossref PubMed Scopus (100) Google Scholar, 11Wagner T.M. Mullally J.E. Fitzpatrick F.A. J. Biol. Chem. 2006; 281: 2598-2604Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 30Moos P.J. Edes K. Cassidy P. Massuda E. Fitzpatrick F.A. J. Biol. Chem. 2003; 278: 745-750Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Immunoblot analysis showed that 5 μm 15d-PGJ2-B alkylated (carbonylated) cellular HDAC1, -2, and -3, but not -8. Formation of an HDAC·15d-PGJ2-B covalent adduct was detectable within 15 min; half-maximal by 30 min; and durable for longer than 180 min (Fig. 1, A and B, and data not shown). Alkylation of intracellular HDACs appears to prefer HDAC2 > HDAC1 > HDAC3; however, alkylation of HDAC8 was not detectable up to 6 h. We also tested the capability of PGD2-biotin, the precursor of 15d-PGJ2-B, in labeling cellular HDAC1. Similar to 15d-PGJ2-B, PGD2-biotin also accumulated HDAC1·PG-biotin adducts (supplemental Fig. 2), but ∼4-fold more slowly (supplemental Fig. 2). This kinetic difference derives from rate-limiting processes, such as dehydration of PGD2-biotin, which yields 15d-PGJ2-B as a terminal metabolite, and temperature-dependent transport of PGJ metabolites into the nucleus (14Narumiya S. Ohno K. Fujiwara M. Fukushima M. J. Pharmacol. Exp. Ther. 1986; 239: 506-511PubMed Google Scholar). PGD2, a prominent eicosanoid from mast cells (31Roberts 2nd, L.J. Sweetman B.J. Prostaglandins. 1985; 30: 383-400Crossref PubMed Scopus (37) Google Scholar) and other hematopoietic cells, helps resolve acute inflammation via mechanisms that involve its transformation into PGJ2, Δ12-PGJ2, and 15d-PGJ2 (32Rajakariar R. Hilliard M. Lawrence T. Trivedi S. Colville-Nash P. Bellingan G. Fitzgerald D. Yaqoob M.M. Gilroy D.W. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 20979-20984Crossref PubMed Scopus (202) Google Scholar). Further investigations suggest that: 1) HDACs react with 15d-PGJ2-B (a bifunctional dienone) better than PGA1-biotin (a mono-functional enone), consistent with precedents (33Uchida K. Shibata T. Chem. Res. Toxicol. 2008; 21: 138-144Crossref PubMed Scopus (126) Google Scholar, 34Gayarre J. Stamatakis K. Renedo M. Pérez-Sala D. FEBS Lett. 2005; 579: 5803-5808Crossref PubMed Scopus (50) Google Scholar) (supplemental Fig. 1A); and 2) that a Michael adduct forms between an HDAC cysteinyl thiol and the electrophilic β-carbon of cyclopentenone PGs (supplemental Fig. 1, B and C). Mammalian class I HDACs and yeast RPD3 have conserved Cys residues. Thus, some other members of this family, besides HDAC1, might be alkylated (carbonylated) by reactive carbonyl species. We found that 15d-PGJ2-B also alkylated RPD3, a class I HDAC homolog in S. cerevisiae (see Fig. 5B and supplemental Fig. 1D). Alkylation of RPD3 and HDAC1, -2, and -3 was concentration-dependent from 1 to 10 μm 15d-PGJ2-B (see Fig. 5B and supplemental Fig. 1D). Covalent modification of HDAC1, -2, and -3, but not HDAC8, occurred uniformly in several cell lines, e.g. HCT116, MCF7, A549, HL-60, and HEK293. Differential alkylation (carbonylation) of some, but not every class I HDAC, implies that their covalent modification is not indiscriminate and may involve particular, conserved cysteinyl thiol residues. Our data (Fig. 1) argue against the cysteine residues corresponding to Cys151 of HDAC1 because these catalytic cysteines are shared by all class I HDACs, including HDAC8. Protein sequence alignment revealed two other cysteine residues in HDAC1, -2, and -3 and RPD3 that we considered candidates for modification (Fig. 2A). These correspond to Cys261 and Cys273 residues of HDAC1, which appear to be surface-accessible, according to homology models (35Wang D.F. Helquist P. Wiech N.L. Wiest O. J. Med. Chem. 2005; 48: 6936-6947Crossref PubMed Scopus (188) Google Scholar). Notably, HDAC8, which was not susceptible to alkylation by 15d-PGJ2-B, has a Leu substituted for Cys at one conserved position and a displaced Cys with a different flanking residue at the other conserved position. The motif surrounding Cys273 of HDAC1 shares appreciable homology with motifs surrounding Cys residues alkylated by cyclopentenone prostaglandins in other proteins, such as the T-loops of the IκB kinase β and LKB1 Ser/Thr kinases, h-Ras, phosphatase and tensin homolog (PTEN), and the p50 and p65 subunits of NFκB. To determine whether any of these cysteine residues reacted with 15d-PGJ2-B, we used site-directed mutagenesis of an HDAC1-FLAG construct to make Cys → Ser mutants, C261S and C273S. Plasmids expressing these HDAC1-FLAG mutants were transfected into HEK293 cells, which were subsequently treated with 5 μm 15d-PGJ2-B. When compared with the wild type HDAC1-FLAG, each of these single-mutant HDAC1-FLAG proteins formed ∼70% less covalent adduct with 15d-PGJ2-B (Fig. 2B, lanes 2 and 3 versus lane 1). Each showed comparable alkylation, consistent with reaction at Cys261 and Cys273. An HDAC1-FLAG double-mutant, C261S/C273S, showed ∼95% less alkylation by 5 μm 15d-PGJ2-B (Fig. 2B, lane 4 versus lane 1), supporting the conclusion that both Cys261 and Cys273 are susceptible to alkylation (carbonylation) by α,β-unsaturated carbonyl compounds. A corresponding set of Cys → Ala mutants gave similar results. There was a low but detectable formation of 15d-PGJ2-B adduct with the C261S/C273S HDAC1 double mutant, ∼5%, implying that other sites may be alkylated to a minor degree. To better understand the functional consequences of HDAC alkylation by RCS, we tested in vitro deacetylase activity of HDAC3·N-CoR1 treated with 4HNE, an enal that can be generated by oxidative stress (36Schneider C. Porter N.A. Brash A.R. J. Biol. Chem. 2008; 283: 15539-15543Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar), and 15d-PGJ2-B. Inhibition of the HDAC3·N-CoR1 complex by these RCS was concentration-dependent and intermediate in potency, relative to TSA and butyrate. Half-maximal inhibition (IC50) required 10 nm TSA, 13 μm 15d-PGJ2-B, 98 μm 4HNE, and 140 μm butyrate (Fig. 3). Maximal inhibition of HDAC3·N-CoR1 by 15d-PGJ2-B and 4HNE is 50 and 70%, respectively. The lack of complete inhibition upon alkylation suggests that inhibition of deacetylase activity is not likely the major consequence of HDAC alkylation yet implies that its modifiable thiolate residues are near the catalytic site or that they are allosterically linked to the catalytic site. Class I HDACs in co-repressor complexes play a major role in transcriptional silencing (18Hassig C.A. Fleischer T.C. Billin A.N. Schreiber S.L. Ayer D.E. Cell. 1997; 89: 341-347Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar, 37Rundlett S.E. Carmen A.A. Kobayashi R. Bavykin S. Turner B.M. Grunstein M. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 14503-14508Crossref PubMed Scopus (514) Google Scholar). Thus, we hypothesized that alkylation and inhibition of HDACs by 15d-PGJ2 would antagonize their co-repressor function in HEK293 cells harboring an STF luciferase reporter with seven LEF/TCF-binding sites (38Xu Q. Wang Y. Dabdoub A. Smallwood P.M. Williams J. Woods C. Kelley M.W. Jiang L. Tasman W. Zhang K. Nathans J. Cell. 2004; 116: 883-895Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar). LEF1 recruits HDAC1 as a co-repressor of transcription in this system. Others have reported that inhibition of HDAC1 by TSA relieves LEF1-mediated repression and converts it to a transcriptional activator (23Billin A.N. Thirlwell H. Ayer D.E. Mol. Cell. Biol. 2000; 20: 6882-6890Crossref PubMed Scopus (189) Google Scholar). If 15d-PGJ2-B or other RCS inhibited HDAC1 in an analogous experiment, LEF1 would be similarly activated, leading to enhanced luciferase expression. Consistent with this hypothesis, luciferase expression rose 20–80-fold as a function of 15d-PGJ2 concentration in HEK293 cells stably expressing an STF reporter construct (Fig. 4A). To determine whether 15d-PGJ2, and related RCS, act independently of other, upstream WNT signaling events, we also used STF3a cells, a cell line that stably expresses both the STF luciferase reporter gene and the wnt3a gene. In these cells, constitutively secreted WNT3a binds to FRZL, its cognate receptor, leading to nuclear accumulation of β-catenin and transactivation of the STF luciferase reporter gene (26McCulloch M.W. Coombs G.S. Banerjee N. Bugni T.S. Cannon K.M. Harper M.K. Veltri C.A. Virshup D.M. Ireland C.M. Bioorg. Med. Chem. 2009; 17: 2189-2198Crossref PubMed Scopus (40) Google Scholar). In conjunction, we used plasmids expressing chimeric MAD·LEF1 genes. The MAD component of the chimera associates with mSin3a in a multiprotein complex that recruits HDAC1 as a co-repressor (Fig. 4C) (18Hassig C.A. Fleischer T.C. Billin A.N. Schreiber S.L. Ayer D.E. Cell. 1997; 89: 341-347Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar). In transfected STF3a cells, the MAD·LEF1 chimer" @default.
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• W2103102045 title "Redox Signaling, Alkylation (Carbonylation) of Conserved Cysteines Inactivates Class I Histone Deacetylases 1, 2, and 3 and Antagonizes Their Transcriptional Repressor Function" @default.
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