Mechanisms of 4Hydroxy2-nonenal Induced Pro and Anti-Apoptotic Signaling

Mechanisms of 4Hydroxy2-nonenal Induced Pro and Anti-Apoptotic Signaling
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  Mechanisms of 4-Hydroxy-2-nonenal Induced Pro- and Anti-Apoptotic Signaling Pankaj Chaudhary , Rajendra Sharma * , Abha Sharma , Rit Vatsyayan , Sushma Yadav , Sharad S. Singhal , Navin Rauniyar , Laszlo Prokai , Sanjay Awasthi , and Yogesh C. Awasthi * Department of Molecular Biology and Immunology, University of North Texas Health ScienceCenter, Fort Worth, Texas 76107 Abstract In recent years, 4-hydroxy-2-nonenal (4-HNE) has emerged as an important second messenger incell cycle signaling. Here we demonstrate that 4-HNE induces signaling for apoptosis via both, theFas mediated extrinsic and the p53 mediated intrinsic pathways in HepG2 cells. 4-HNE inducesFas-mediated DISC independent apoptosis pathway by activating ASK1, JNK and caspase-3. Inparallel treatment of 4-HNE to HepG2 cells also induces apoptosis by p53 pathway throughactivation of Bax, p21, JNK, and caspase-3. Exposure of HepG2 cells to 4-HNE leads to theactivation of both Fas and Daxx, promotes the export of Daxx from the nucleus to cytoplasm andfacilitates Fas-Daxx binding. Depletion of Daxx by siRNA results in potentiation of apoptosisindicating that Fas-Daxx binding in fact is inhibitory to Fas mediated apoptosis in cells. 4-HNE-induced translocation of Daxx is also accompanied by the activation and nuclear accumulation of HSF1 and up-regulation of heat shock protein Hsp70. All these effects of 4-HNE in cells can beattenuated by ectopic expression of hGSTA4-4, the isozyme of glutathione S  -transferase with highactivity for 4-HNE. Through immunoprecipitation and liquid chromatography–tandem massspectrometry, we have demonstrated covalent binding of 4-HNE to Daxx. We also demonstratethat 4-HNE modification induces phosphorylation of Daxx at Ser668 and Ser671 to facilitate itscytoplasmic export. These results indicate that while 4-HNE exhibits toxicity through severalmechanisms, in parallel it evokes signaling for defense mechanisms to self regulate its toxicity andcan simultaneously affect multiple signaling pathways through its interactions with membranereceptors and transcription factors/ repressors.Reactive oxygen species (ROS) produced during exposure of cells to UV radiation, heatshock, or xenobiotics and during metabolic processes cause the oxidation of polyunsaturatedfatty acids in membrane lipid bilayers that are one of the early targets of ROS. Previousstudies have indicated that the exposure of cells to even minimal transient stress causessubstantial lipid peroxidation (LPO) (1,2). These studies show that transient exposure of cells to UV, H 2 O 2 , or oxidants leads to a significant (~50%) rise in the levels of 4-hydroxy-2-nonenal (4-HNE) which is a stable end-product of LPO. 4-HNE has been widelyimplicated in the mechanisms of oxidant toxicity and also in cell cycle signaling (3–9) butmost of the associated mechanisms are not completely understood. Liver being the majorsite for metabolism, and biotransformation of xenobiotics is constantly exposed to ROS. Inthe event of insufficient levels of defense mechanisms such as free-radical scavengers or * To either of whom the correspondence and reprint requests should be addressed: Department of Molecular Biology and Immunology,University of North Texas Health Science Center, Fort Worth, Texas 76107. Tel: 817-735-2366; Fax: 817-735-2118;,, or SUPPORTING INFORMATION AVAILABLE Additional Materials and Methods, and Figures S1–S5 as described in the text. This material is available free of charge via the Internetat NIH Public Access Author Manuscript  Biochemistry . Author manuscript; available in PMC 2011 July 27. Published in final edited form as: Biochemistry  . 2010 July 27; 49(29): 62636275. doi:10.1021/bi100517x. N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t    antioxidants, increasing levels of lipid hydroperoxides and peroxides can be produced inliver by self-perpetuating chain reactions resulting in the formation of 4-HNE that can exerttoxicity through interactions with cellular macromolecules, including proteins, lipids, andnucleic acids. For example, chronic alcohol consumption (10), high-iron diet (11), high-fatdiet (12), or exposure to hepatotoxic agents like CCl 4  markedly elevate the intracellularconcentration of 4-HNE from its basal constitutive levels and damage hepatocytes and theliver (13,14). 4-HNE is also believed to be involved in the mechanisms of diseases such asatherosclerosis (15,16), diabetes (17), Alzheimer’s disease (18,19), Parkinson's disease (20),cataract (21), and cancer (22,23).In recent past (1,2,6–9), studies in our laboratories have clearly shown that signaling forapoptosis by many oxidants including superoxide anion generated by xanthine-xanthineoxidase system is mediated through 4-HNE and it could be inhibited by acceleratingdisposal of 4-HNE by forced over expression of GSTA4-4 which is highly specific for 4-HNE but is not involved in the detoxification of ROS such as H 2 O 2 , O 2• − . A multitude of studies by other investigators (3–5,24), and in our laboratories indicate a global role of 4-HNE in modulation of cellular processes. Studies in our lab have shown that the signalingfor proliferation, transformation, apoptosis, and differentiation is associated with alterationsin the intracellular levels of 4-HNE in a wide variety of cells (9,25–28). These findings posean intriguing question as to how 4-HNE is able to exert such a multitude of effects oncellular processes.During the present studies we have addressed this question by investigating the role of 4-HNE mediated signaling in the pathways associated with the regulation of programmed celldeath in a liver derived cell line HepG2. Specifically, we have investigated the effect of increased 4-HNE concentrations in cells by direct exposure or by oxidant treatment ondifferent pathways leading to apoptosis. Conversely, we have evaluated the role of 4-HNE insignaling of these pathways by lowering its intracellular concentration through thetransfection of cells with hGSTA4 . We have also studied the interactions of 4-HNE withsome of the key proteins involved in these pathways. Furthermore, to examine the in vivo significance of these findings we have also studied some of these effects of 4-HNE in theliver tissues of Gsta4  null mice where 4-HNE levels are consistently maintained at highlevels due to its impaired disposition (29). The results of these studies show that 4-HNEcauses toxicity to HepG2 cells via necrosis and apoptosis induced by more than onepathway. These findings integrate the mechanisms for the multifarious effects of 4-HNE oncellular processes suggesting that 4-HNE through direct interactions with membranereceptors, transcription factors, and transcription repressors regulates trafficking, and thesignaling functions of key proteins to affect various cellular processes. MATERIALS AND METHODS Materials 4-Hydroxynonenal was purchased from Cayman Chemical (Ann Arbor, MI). Bradfordreagent, bis-acrylamide, and SDS for SDS-PAGE were obtained from BioRad (Hercules,CA). The apoptosis detection system (CaspACE FITC-VAD-FMK in situ  marker) waspurchased from Promega Inc. (Madison, WI). The cytoplasmic and nuclear proteinextraction kit was acquired from Imgenex Co. (San Diego, CA), protein A/G-agarose fromSanta Cruz Biotechnology (Santa Cruz, CA), JNK inhibitor SP6000125 from A–G Scientific(San Deigo, CA), and Western blot stripping buffer from Pierce Co. (Rockford, IL). Allother reagents and chemicals were purchased from Sigma-Aldrich (St. Louis, MO). The cellculture medium RPMI-1640, geneticin (G418), Lipofectamine 2000 transfection reagent andfetal bovine serum were from GIBCO (Invitrogen, Carlsbad, CA). Chaudhary et al.Page 2  Biochemistry . Author manuscript; available in PMC 2011 July 27. N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t    Cell lines and Culture Conditions The HepG2 human hepatocarcinoma cells purchased from the American Type CultureCollection were cultured in RPMI-1640 supplemented with 10% fetal bovine serum, 1% of astock solution containing 10,000 IU/mL penicillin and 10 mg/mL streptomycin in anincubator at 37°C under a humidified atmosphere containing 5% CO 2 . Preparation of cell extracts and Western blot analysis Cells were collected, washed with cold PBS and then incubated in 100 µL of RIPA lysisbuffer (50 mM Tris-HCl, pH 7.5; 1% NP-40; 150 mM NaCl; 1 mg ml − 1  aprotinin; 1 mgml − 1  leupeptin; 1 mM Na 3 VO 4 ; 1 mM NaF) at 4°C for 30 min. Cell debris was removed bycentrifugation at 12,000 g  for 10 min at 4°C. Protein concentrations were determined byBradford assay (30) as described in standard protocol. Cell extracts were separated on SDSpolyacrylamide gels (4–20%), and transferred onto nitrocellulose (Bio-Rad). Membraneswere blocked with 5% fat-free milk at room temperature for 60 min, and incubatedovernight at 4°C with the appropriate primary antibody in 5% milk in Tris-buffered saline(TBS) containing 50 mM NaF and 0.05% Tween 20. After three times washing with T-TBS(Tris-buffered saline containing 0.05% Tween 20), the membrane was incubated with theappropriate secondary antibody at room temperature for 2 h. After washing again with T-TBS, the membrane was treated with Super signal ‘West Pico’ chemiluminescent reagent(Pierce, Rockford, IL) as per manufacturer's instructions, and exposed to Hyperfilm ECLfilm (Amersham) at room temperature. Isolation of nuclear and cytoplasmic fractions wasachieved by Imgenex nuclear extraction kit as per the manufacturer’s instructions (Imgenex,San Deigo, CA). Stable transfection with pTarget and hGSTA4 HepG2 cells at a density of 5 × 10 5  cells per 100 mm Petri dish were plated for thetransfection. Petri dishes having >50% confluent cells were used for the transfection. Thecells were transfected with 24 µg of either empty pTarget-T vector (VT) or the pTargetvector with the open reading frame (ORF) of the hGSTA4  sequence ( hGSTA4-Tr  ), usingLipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) as per the manufacturer’sinstructions. Transfection of Daxx siRNA in HepG2 cells Small interfering RNA (siRNA) transfection experiments against Daxx were performedusing double-stranded RNA synthesized by Dharmacon (ON-TARGET Plus  SMARTpool,Dharmacon, Chicago, IL). Briefly, HepG2 cells (2 × 10 5  cells per well) were plated in a six-well tissue culture plate, in 2 mL normal growth medium supplemented with FBS. Cellswere cultured at 37°C until 60–80% confluency. For each transfection, 100 nM double-stranded non-targeting control siRNA (Dharmacon, used as control), or Daxx-specificsiRNA were transfected into HepG2 cells using DharmaFECT 4 transfection reagent(Dharmacon) according to the manufacturer’s protocol. Cells were harvested at appropriatetime points and the silencing of Daxx was examined by Western blotting. Immunofluorescence studies HepG2 cells were grown to 50% confluence on glass cover slips in 12-well plates. The cellswere exposed to 20 µM 4-HNE. Untreated cells remained as controls. Treated and untreatedcells were incubated for 2 h, washed twice with ice-cold PBS (pH 7.4), fixed with 4%paraformaldehyde for 30 min and then permeabilized with 0.1% Triton X-100 for 30 min.The slides were then washed with PBS, incubated with 5% goat serum in PBS for 2 h, andthen incubated with anti-Daxx or anti-HSF1 antibodies (Santa Cruz Biotechnology, SantaCruz, CA) diluted 1:50 in PBS containing 1% goat serum for overnight at 4°C temperature. Chaudhary et al.Page 3  Biochemistry . Author manuscript; available in PMC 2011 July 27. N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t    After washing with ice cold PBS, the cover slips were incubated with FITC-labeled goatanti-rabbit immunoglobulin G (Southern Biotech, USA) diluted 1:200 in PBS containing 1%goat serum for 2 h at room temperature in dark. The cover slips were then washed threetimes with ice cold PBS and mounted on glass slides with 20 µL of VectaShield mediumcontaining DAPI (1.5 µg/mL) (Vector Laboratories, Inc., USA). The slides were examinedusing LSM 510 Meta confocal system equipped with an inverted microscope (AxioObserver Z1, Carl Zeiss). In situ caspase-3 assay for Apoptosis Cells (2 × 10 4 ) were treated with 0–40 µM 4-HNE or with 250 ng/mL Fas agonistic CH11antibodies, that are known to induce apoptosis, for 2 h at 37°C. Apoptotic cells weredetected by staining with in situ  marker (10 µM, CaspACE FITC-VAD-FMK, Promega) for30 min in the dark. The slides were fixed with 4% paraformaldehyde for 30 min, rinsed withPBS twice, mounted in a medium containing 1.5 µg/mL DAPI and observed under OlympusAX70 fluorescence microscope. Chromatin Immunoprecipitation (ChIP) assay To determine whether after nuclear translocation HSF1 binds to  Hsp70  promoter, ChIPassay was performed using the ChIP-IT kit from Active Motif (Carlsbad, CA) following themanufacturer's instructions. Briefly, 5 × 10 5  cells were grown and treated with 20 µM 4-HNE for 2 h and fixed with 1% formaldehyde. Cells were washed with PBS followed by theaddition of glycine stop solution and washing with PBS. The cells were collected andresuspended in lysis buffer, incubated for 10 min on ice, vortexed for 10s, and centrifugedfor 10 min at 4,000 g  at 4°C. The DNA pellet was resuspended in shearing buffer, sonicatedand the sheared DNA sample was centrifuged. The resultant supernatant was incubated withpositive control IgG as well as negative control IgG (provided by Active Motif) and anti-HSF1 IgG (Santa Cruz, CA) overnight at 4°C. Protein G beads were added to the reactionmixtures and incubated for 2 h at 4°C. The beads were pelleted by centrifugation followedby washing, and eluted with 100 µL of ChIP elution buffer. Eluted DNA samples werepurified using the DNA purification mini columns and amplified by PCR using the controlprimers and negative control primers (provided by Active Motif) and hHsp70 primers. PCRproducts were analyzed by running on 1% agarose gels. Preparation of 4-HNE-Daxx adduct and identification of 4-HNE binding sites by liquidchromatography–tandem mass spectrometry (LC–MS/MS) 4-HNE adducts of purified bacterially expressed human Daxx (1 mg/mL) were prepared byreacting with 2 mM 4-HNE in 0.1 M phosphate buffer, pH 7.4, at 37°C for 2 h. Theenrichment of 4-HNE-modified peptides from the proteolytic digest of Daxx wasaccomplished by using solid-phase hydrazide (SPH) reagent as described previously (31)and analyzed by LC–MS/MS analyses (32,33). LC–MS/MS was performed on a hybridlinear ion trap-FTICR (7-Tesla) mass spectrometer (LTQ-FT, Thermo Finnigan, San Jose,CA) equipped with a nanoelectrospray ionization source and operated with the Xcalibur(version 2.2) and Tune Plus (version 2.2) data acquisition software. MS/MS data generatedby data dependent acquisition via the LTQ-FT were extracted by BioWorks version 3.3 andsearched against a composite IPI human (version 3.47, number of entries is 144164 × 2)protein sequence database containing both forward and randomized sequences using theMascot v 2.2 (Matrix Science, Boston, MA) search algorithm. (34,35) MS/MS spectra of 4-HNE modified peptides were visually inspected to verify peptide fragment ions. The MS-Product module of Protein Prospector ( was used to calculate them/z values of b- and y-type ions of 4-HNE-modified Daxx tryptic peptides, obtained duringCID-MS/MS, where His residues were replaced with 4-HNE-His Michael adducts (M +156). Chaudhary et al.Page 4  Biochemistry . Author manuscript; available in PMC 2011 July 27. N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t    Statistical Analysis The data are expressed as the mean ± SD for each group. The statistical significance wasdetermined by Student's t   test and was set at  p  < 0.05. RESULTS 4-HNE causes apoptosis and necrosis in HepG2 cells The cytotoxicity of 4-HNE to HepG2 cells was evaluated by MTT assay and apoptosismeasured by flow cytometry, caspase activation, and PARP cleavage. In MTT assay (seesupporting information), 4-HNE concentrations ranging from 10 to 100 µM graduallydecreased cell viability corresponding to an IC 50  value of 53 ± 2.39 µM ( n  = 8). Based onthese results, 4-HNE concentrations of 5–40 µM were used to examine its effect onapoptotic signaling in HepG2 cells. 4-HNE-induced proteolytic cleavage of caspase-3 andPARP was also monitored. In the effector stage of apoptosis, caspase-3 is activated by itsproteolytic cleavage into 17 and 12 kDa fragments. Like wise, poly (ADP-ribose)polymerase (PARP), which is normally involved in DNA repair, DNA stability, and othercellular events, is cleaved by members of the caspase family during early apoptosis. Resultspresented in Figure 1A showed that 4-HNE caused a dose dependent increase in 17 kDafragment from the procaspase-3 and that of the 89 kDa cleavage product from PARP. 4-HNE-induced apoptosis in HepG2 cells was further analyzed by flow cytometry. Resultspresented in Figure 1B showed that after treatment with different concentrations of 4-HNEranging from 0–100 µM for 2 h, the viability of cells decreased from 86.5% to 28.3% withan increase in the percent of late apoptotic cells from 9.7 to 16.1% in a dose dependentmanner. A significant increase in necrotic cell population i.e. 31.8% and 55.4%, wasobserved in cells treated with 80 and 100 µM of 4-HNE respectively (Figure 1C). Theseresults indicated that initial response to sub-lethal doses of 4-HNE (5–40 µM) treatmentpredominantly caused apoptotic cell death that ultimately proceeded to necrosis of HepG2cells at lethal 4-HNE concentrations (80–100 µM). Over-expression of hGSTA4-4 inhibits 4-HNE induced apoptosis HepG2 cells were stably transfected with hGSTA4  and over expression of hGSTA4-4protein was confirmed by the results of Western blot analysis (Figure 2A). GST activitytowards 4-HNE was found to be enhanced in hGSTA4  transfected cells along with theexpected decrease in the constitutive 4-HNE levels (data not presented). 4-HNE-inducedapoptosis in the empty vector and hGSTA4  transfected cells was analyzed by usingCaspACE™ FITC-VAD-FMK in situ  marker that binds to the cleaved caspase-3. Results of these experiments showed that the hGSTA4  transfected cells acquired significant resistanceto 4-HNE-induced apoptosis as compared to the empty vector transfected HepG2 cells(Figure 2B upper panel). hGSTA4  transfected cells also acquired significant resistance toDoxorubicin (DOX)-induced apoptosis (Figure 2B lower panel). Since DOX-inducedapoptosis has been attributed to generation of ROS, these results suggest the role of 4-HNEin the mechanism of apoptosis caused by oxidants in general (6, 7). 4-HNE and Fas mediated apoptosis4-HNE activates Fas— To elucidate the mechanism of 4-HNE-induced apoptosis inHepG2 cells, we analyzed first the effect of 4-HNE on the expression of Fas. Results of Western blot analyses (Figure S1A and B in supporting information) indicated that 4-HNEcaused a time, and dose dependent induction of Fas in HepG2 cells. These results wereconsistent with the previously reported induction of Fas by 4-HNE in HLE B-3, Jurkat, andCRL2571 cells (26, 28) indicating that 4-HNE mediated induction of Fas is not limited to aspecific cell types. Chaudhary et al.Page 5  Biochemistry . Author manuscript; available in PMC 2011 July 27. N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t  
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