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4-Hydroxynonenal induces p53-mediated apoptosis in retinal pigment epithelial cells

4-Hydroxynonenal induces p53-mediated apoptosis in retinal pigment epithelial cells
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  4-Hydroxynonenal induces p53-mediated apoptosis in retinalpigment epithelial cells Abha Sharma a, Rajendra Sharma a, Pankaj Chaudhary a, Rit Vatsyayan a, Virginia Pearce b, Prince V.S. Jeyabal c, Piotr Zimniak d, Sanjay Awasthi a, and Yogesh C. Awasthi a,*a  Department of Molecular Biology and Immunology, RES 416G, University of North Texas Health ScienceCenter, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107, USA b  Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, TX, USA c  Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX,USA d  Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare System, Little Rock, AR, USA Abstract 4-Hydroxynonenal (4-HNE) has been suggested to be involved in stress-induced signaling forapoptosis. In present studies, we have examined the effects of 4-HNE on the intrinsic apoptoticpathway associated with p53 in human retinal pigment epithelial (RPE and ARPE-19) cells. Ourresults show that 4-HNE causes induction, phosphorylation, and nuclear accumulation of p53 whichis accompanied with down regulation of MDM2, activation of the pro-apoptotic p53 target genesviz. p21 and Bax, JNK, caspase3, and onset of apoptosis in treated RPE cells. Reduced expressionof p53 by an efficient silencing of the p53 gene resulted in a significant resistance of these cells to4-HNE-induced cell death. The effects of 4-HNE on the expression and functions of p53 are blockedin GSTA4-4 over expressing cells indicating that 4-HNE-induced, p53-mediated signaling forapoptosis is regulated by GSTs. Our results also show that the induction of p53 in tissues of  mGsta4  (-/-) mice correlate with elevated levels of 4-HNE due to its impaired metabolism. Together,these studies suggest that 4-HNE is involved in p53-mediated signaling in in vitro  cell cultures aswell as in vivo  that can be regulated by GSTs. Keywords 4-Hydroxynonenal; Oxidative stress; p53; Glutathione S  -transferase; Lipid peroxidation; Apoptosis;Retinal pigment epithelial (RPE) cells Introduction Exposure of cells and tissues to xenobiotics or physico-chemical sources of oxidative stress(e.g. UV) causes the generation of reactive oxygen species (ROS), which in turn can causedamage by interacting with cellular macromolecules such as DNA, proteins, and lipids, as wellas by forming secondary toxic products. ROS-initiated membrane lipid peroxidation (LPO) ishighly damaging to cells because a single oxidative event can start a chain reaction and lead *Corresponding author. Fax: +1 817 735 2118.  E-mail address : (Y.C. Awasthi).. NIH Public Access Author Manuscript  Arch Biochem Biophys . Author manuscript; available in PMC 2009 December 15. Published in final edited form as:  Arch Biochem Biophys . 2008 December 15; 480(2): 8594. doi:10.1016/ 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    to the formation of large amounts of LPO products including toxic electrophiles such as 4-hydroxynonenal (4-HNE) [1]. In recent years, 4-HNE has been shown to be an importantsecond messenger involved in signaling for cell cycle arrest, differentiation, apoptosis andregulation of the expression of a multitude of genes including p53, in cells of diverse srcin[1-3]. P53 plays an important role in apoptosis, growth arrest, genomic stability, cellsenescence, and differentiation. It is normally kept at a low concentration in cells by itsrelatively short half-life. Some cells may also have a latent form of p53 which is inactive fortranscription [4-6]. Diverse cellular and stressful events such as DNA damage, hypoxia, andoxidative stress activate the p53 gene by causing it to accumulate rapidly through aposttranscriptional mechanism, generally in a proportion to the extent of DNA damage [4,7].In addition to induction through stress signals, increased quantities of p53 protein in cells arefound in cells modified by T antigen and E1a, the gene products encoded by the DNA virusesSV40 and adenovirus, respectively [8]. While the SV40 Tag is known to bind to the p53 protein[9], and extend the lifespan of human cells [10], more recent studies have demonstrated afunctional p53 in the SV40-transformed human [9] and even the murine [11] cells.Once activated by phosphorylation [12-14], the modified and stable p53 can induce up-regulation of the downstream target genes, p21 and Bax [15-18]. Expression of the p21 geneproduct inhibits the cyclin-dependent kinase activity and arrests cell cycle progression whilep53-dependent Bax synthesis induces apoptosis by binding to Bcl-2 and antagonizing its non-apoptotic ability. Since generation of 4-HNE has been suggested to be a common denominatorin mechanisms of apoptosis caused by diverse forms of oxidative stress [19,20], it is likely thatit would also affect the expression and activation of p53. This idea is supported by our recentstudies showing that alterations in the intracellular levels of 4-HNE in HLE B-3 cells affectthe expression of a multitude of genes including p53 [21,22]. Thus, it is possible that 4-HNE-mediated activation of p53 may be one of the mechanisms responsible for 4-HNE-inducedapoptosis reported in many cell types [19,23-27].Retina is particularly susceptible to oxidative stress because not only it is bombarded constantlyby ROS-producing UV and high-energy visible light [28], but also because retinal pigmentepithelial (RPE) cells maintain and support the photoreceptors by phagocytosis and degradationof the photoreceptor outer segment membranes which are rich in polyunsaturated fatty acids[29-31]. It has been suggested that LPO products contribute to retinal pigment epithelialdysfunction, initiating retinal degenerative disorders including age-related maculardegeneration (ARMD) which is the leading cause of blindness in the developed world [32].Present studies were designed to study the effects of 4-HNE on the expression and activationof p53 in RPE cells focusing on the p53-mediated intrinsic pathway for apoptosis. Glutathione S  -transferase A4-4 (GSTA4-4)-mediated metabolism of 4-HNE is one of the majordeterminants of the intracellular concentration of 4-HNE [21,22,33]. Therefore, we have alsoexamined the possible role of GSTA4-4 in regulation of 4-HNE-induced, p53-mediatedapoptosis in RPE cells. For these studies, we have chosen RPE cells of human fetal srcin andARPE-19 cells developed from the retina of adult young male. Results of these studies indicatethat in these cells 4-HNE causes activation, phosphorylation, and enhanced nuclearaccumulation of p53, accompanied with activation of the signaling components involved inp53-mediated apoptosis. Over-expression of either human GSTA4-4 or the correspondingmurine isozyme mGsta4-4 as well as the silencing of cellular p53 blocks these effects of 4-HNE. Materials and methods Chemicals Dulbecco's modified Eagle's medium (DMEM), penicillin-streptomycin solution (P/S),phosphate buffered saline (PBS), fetal bovine serum (FBS), HEPES, trypsin, and MEM non- Sharma et al.Page 2  Arch Biochem Biophys . Author manuscript; available in PMC 2009 December 15. 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    essential amino acid solution were purchased from Gibco (Grand Island, NY). 4-HNE waspurchased from Cayman Chemical (Ann Arbor, MI). Reagents for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transblotting were purchased from Bio-Rad (Hercules, CA). RIPA lysis buffer was obtained from Santa Cruz Biotechnology (SantaCruz, CA). The Western blot stripping buffer was from Pierce (Rockford, IL). The apoptosisdetection system (CaspACE™ FITC-VAD-FMK in situ  marker) was purchased from PromegaInc. (Madison, WI). All other chemicals and reagents were obtained from Sigma-Aldrich (St.Louis, MO). Antibodies Antibodies against p53 (DO-1, sc-126, mouse monoclonal), JNK1 (sc-571, polyclonal), p-JNK(G-7, sc-6254, monoclonal) p21 (sc-6246, monoclonal), Bax (N-20, sc-493, polyclonal)caspase3 (sc-7148, polyclonal) and MDM2 (sc-965, monoclonal), Histone H1 (sc-8030,monoclonal), GAPDH (sc-32233, monoclonal) were obtained from Santa Cruz Biotechnology(Santa Cruz, CA) while those against phosphorylated p53 (ser15, 9284S, polyclonal) wereobtained from Cell Signaling Technology, Inc. (MA). Polyclonal antibodies developed againstrecombinant hGSTA4-4 in chicken and mGsta4-4 in rabbit have been characterized anddescribed by us previously [33,34]. Horseradish peroxidase (HRP)-conjugated secondaryantibodies as well those against GAPDH were purchased from Southern Biotech (Birmingham,AL). Cell lines The simian virus SV40-transformed human fetal male RPE 28 cells (Coriell Institute, Camden,NJ) which exhibit epitheloid morphology and retain physiological functions characteristic of the primary human RPE cells were chosen as a suitable model for investigating the effect of low levels of oxidative stress [31]. The ARPE-19 (ATCC CRL-2302) is a spontaneously arisingretinal pigment epithelia cell line derived from the normal eyes of a young male. Cells werecultured in their standard medium containing 10% fetal bovine serum and antibiotics in ahumidified incubator at 37 °C in 5% CO 2  atmosphere. Both cell lines were trypsinized andpassaged every 3-4 days. Cell viability The sensitivity of the RPE and ARPE-19 cells to 4-HNE was measured by the MTT assay asdescribed by Mosmann [35] with slight modifications. After determining the number of viablecells in an aliquot of a log-phase culture by counting trypan-blue excluding cells in ahemocytometer, the cells were re-suspended in the regular complete medium. About 2 × 10 4 cells in an aliquot of 190 μ l of full serum medium were seeded in 96-well flat bottomedmicrotiter plates for 24 h to allow attachment to the culture plates. After confirming cellattachment, the cells were incubated for 18 h in fresh serum-free medium to avoid anyinteraction between serum proteins and 4-HNE. No significant apoptosis was observed inserum-deprived conditions. The next day, 10 μ l of PBS containing various amounts of 4-HNEwas added. Eight replicate wells were used for each concentration of 4-HNE in these studies.After 24 h incubation of the plates at 37 °C, 10 μ l of MTT solution (5 mg/ml in PBS) was addedto each well, and the plates incubated for an additional 4 h at 37 °C. The plates were thencentrifuged at 2000 g  for 10 min and the medium aspirated from each well. 100 μ l of dimethylsulfoxide (DMSO) was added to each well and the intracellular formazan dye crystalswere dissolved by shaking the plates at room temperature for at least 30 min. The absorbanceof formazan at 562 nm was measured using a microplate reader (El × 808 BioTek Instruments,Inc). A dose-response curve was plotted and the concentration of 4-HNE resulting in a 50%decrease in formazan formation was calculated as the IC 50  value of 4-HNE. Sharma et al.Page 3  Arch Biochem Biophys . Author manuscript; available in PMC 2009 December 15. 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    Exposure of cells to 4-HNE for signaling studies RPE and ARPE-19 cells were plated at a density of 4 × 10 5  per 8.0 ml of complete medium in100 mm dishes overnight. To investigate the effects of 4-HNE in p53-mediated cellularsignaling, we cultured the cells overnight in serum-free conditions as above and then incubatedthem with different concentrations (0-75 μ M) of 4-HNE prepared in phosphate buffered saline(PBS) for a period of 2 h. Preparation of cell and tissue extracts Cells treated as above were thereafter collected, washed with PBS and re-suspended in radio-immunoprecipitation assay (RIPA) lysis buffer, containing 1 × PBS (pH 7.4), 1% Nonidet-P-40 (NP-40), 0.5% sodium deoxycholate, 0.1% sodium dodecyl suphate, 1 mMphenylmethanesulfonyl fluoride (PMSF), and 2 μ g/ml of pepstatin. After sonicating three timesfor 5 s, the lysates were centrifuged at 14,000 rpm for 15 min and the supernatants collected.Brain, heart, lung, kidney, liver, and eye from wild-type and mGsta4  knockout mice werequickly removed after they had been sacrificed and immediately transferred to -80 °C untilthey were to be used. After washing with 20 mM potassium phosphate buffer (pH 7.0)containing 1.4 mM β -mercaptoethanol, the tissue samples were homogenized in ice-cold RIPAbuffer containing PMSF and incubated on ice for at least 30 min. Homogenates werecentrifuged at 14,000 rpm for 15 min and the extracts collected as supernatants were used foranalyses. Protein concentrations were determined by the Bradford assay [36]. Western blot analysis Total cell extracts, containing 25-60 μ g protein were separated by SDS-PAGE (4-20% gels)and transferred onto a nitrocellulose membrane (Bio-Rad). 16% gels were used for the detectionof caspase3. After blocking the membrane with 5% non-fat dry milk in Tris-buffered saline(TBS) at room temperature for 1 h, it was incubated overnight at 4 °C with the appropriateprimary antibody in blocking buffer. After washing with TBS, the membrane was incubatedwith the appropriate secondary antibody at room temperature for 1 h. The membrane was againwashed with TBS, and immunoblots were developed with the ECL (chemiluminescence)reagents from Pierce according to the manufacturer's instructions. For JNK and p-JNKimmunoblot detection, the procedure was slightly modified in that the membranes wereblocked with 1% non-fat milk and 1% BSA instead of the blocking buffer containing 5% non-fat milk. Also, the primary JNK antibody was incubated in Tris-buffered saline (TBS)containing 1% milk, 1% BSA, 50 mM NaF and 0.05% Tween 20 (T-TBS). Transient transfection with GSTA4 We carried out transient transfection in RPE cells using 10 μ g of either empty pTarget-T vector(VT) or the pTarget vector with the open reading frame (ORF) of the restored Kozak  hGSTA4  sequence ( hGSTA4-Tr  ), using Lipofectamine PLUS reagents (Invitrogen, Carlsbad,CA) as per the manufacturer's instructions. Similarly, ARPE-19 cells were transientlytransfected with the mouse pRC/CMV mGsta4  [37] and with the vector alone. For 4-HNEexposure experiments, GSTA4  transiently transfected RPE and ARPE-19 cells were treated for1 h with 20 μ M 4-HNE in serum-free medium and cell lysates prepared again as describedabove. Silencing of p53expression using siRNA The p53 siRNA transfection was essentially carried out according to manufacturer'sinstructions. P53 si RNA (h) (sc-29435) and control si RNA (sc-37007) were obtained fromSanta Cruz Biotechnology (Santa Cruz, CA) to transfect RPE cells. Briefly, RPE cells (2 ×10 5  cells per well) were plated in a six-well tissue culture plate, in 2 ml of antibiotic free growth Sharma et al.Page 4  Arch Biochem Biophys . Author manuscript; available in PMC 2009 December 15. 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    medium supplemented with FBS. Cells were cultured at 37 °C until 60-80% confluency. Foreach transfection, 50 pmol of siRNA was diluted with 100 μ l siRNA transfection medium(solution A) and 8 μ l of siRNA transfection reagent was diluted with 100 μ l siRNA transfectionmedium (solution B). Solution A was directly added to solution B, mixed gently and the mixturewas incubated for 30 min at room temperature. Cells were washed with 2 ml of siRNAtransfection medium and 0.8 ml of siRNA transfection medium was added to the mixture of the solutions A and B, gently mixed and overlaid onto the washed cells and incubated for 24h at 37 °C. After 24 h, 2 ml of fresh normal medium was added to each well and cells werefurther incubated for 48 h. Control cells were treated in a similar manner with a mixture of scrambled siRNA. Cells were harvested at appropriate time points and the silencing of p53was examined. In situ detection of apoptosis ARPE-19 (2 × 10 4 ) cells were plated in complete medium onto chamber slides a day beforethe experiment. On the day of the experiment, the cells were assessed for their confluency(60-80%) and starved of serum for at least 2 h before treatment with different concentrations(0-50 μ M) of 4-HNE in serum-free medium for 2 h at 37 °C. About 1 μ  10 5  RPE cells (VT and hGSTA4-Tr  ) were treated with 20 μ M 4-HNE in serum-free medium for 1 h at 37 °C. Apoptoticcells were detected by staining with 10 μ M CaspACE™ FITC-VAD-FMK in situ  marker for30 min in the dark. After rinsing twice with PBS, the slides were fixed with 4%paraformaldehyde for 1 h, mounted in a medium containing DAPI (1.5 μ g/ml), and observedunder the fluorescence microscope (Olympus, Japan). Determination of 4-HNE levels Intracellular 4-HNE levels in RPE cells were measured spectro-photometrically by using theBiotech LPO-586 228 kit (Oxis International, Portland, OR) as per the manufacturer'sinstructions as well as by the HPLC method described below. Empty vector- and hGSTA4 -transfected RPE cells were sus pended in ice and sonicated (3×, 10 s, 30 W) on ice. Cellularproteins were precipitated with ice-cold 70% perchloric acid (1 ml). After centrifuging at10000 g  for 10 min, the supernatants were extracted with 2 ml of dichloromethane (HPLCgrade) by gentle vortexing. The extracts were once again centrifuged at 1500 rpm for 10 min.The organic layer was collected, dried under nitrogen, re-suspended in 100 μ l ethanol andfiltered through nylon 66 filters (Micron Separations Inc. NY). The Beckman Coulter SystemGold HPLC equipment connected to a Beckman 168 Photo Diode Array (PDA) detector andthe Beckman Ultrasphere (5 μ m, 4.6 × 25 cm) column was used for HPLC analysis of thefiltrate, using 70% sodium phosphate, pH 2.6, 30% acetonitrile as the mobile phase. Thecolumn eluate was monitored for 4-HNE at 202 and 224 nm [38] and peaks were analyzed byBeckman 32 Karat software. 4-HNE concentrations of the samples were quantified by using acalibration graph prepared by HPLC analysis of standard 4-HNE concentrations (0-1000 pmol)plotted against peak areas. Results 4-HNE is toxic to RPE/ARPE-19 cells Because retina is rich in polyunsaturated fatty acids and LPO is believed to be involved in theetiology of retinopathy [39], we first determined the cytotoxicity of 4-HNE to RPE andARPE-19 cells in cultures which was estimated from the percentage of cells surviving theexposure of varying concentrations of 4-HNE for 2 h. Results of these experiments showeddecrease in the cell viability with increasing concentrations of 4-HNE (Fig. 1). The IC 50  values for 4-HNE for the RPE and ARPE-19 cells were found to be 52 ± 1.4, and 46 ± 3.2 μ M ( n  =8), respectively. Sharma et al.Page 5  Arch Biochem Biophys . Author manuscript; available in PMC 2009 December 15. 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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