Automobiles

8 pages
4 views

Activation of p53 and the pro-apoptotic p53 target gene PUMA during depolarization-induced apoptosis of chromaffin cells

Please download to get full document.

View again

of 8
All materials on our website are shared by users. If you have any questions about copyright issues, please report us to resolve them. We are always happy to assist you.
Share
Description
Activation of p53 and the pro-apoptotic p53 target gene PUMA during depolarization-induced apoptosis of chromaffin cells
Transcript
  Activation of p53 and the pro-apoptotic p53 target gene PUMA duringdepolarization-induced apoptosis of chromaffin cells M. Gomez-Lazaro a  , M.F. Galindo a  , F.J. Fernandez-Gomez a  , J.H.M. Prehn  b , J. Jorda´n a, * a   Departamento de Ciencias Me´dicas, Facultad de Medicina, Universidad de Castilla-La Mancha, Avda. Almansa, s/n, 02006 Albacete, Spain  b  Department of Physiology and RCSI Neuroscience Research Centre, Royal College of Surgeons in Ireland, 123 St Stephen’s Green, Dublin 2, Ireland  Received 10 May 2005; revised 8 July 2005; accepted 14 July 2005Available online 19 August 2005 Abstract The pathogenesis of non-glutamatergic, depolarization-induced cell death is still enigmatic. Recently, we have shown that veratridineinduces apoptosis in chromaffin cells, and we have demonstrated protective effects of antioxidants in this system, suggesting a role for Na + channels and oxidative stress in depolarization-induced cell death. We examined the possible contribution of p53, a transcription factor that has a major role in determining cell fate, and the mitochondrial apoptosis pathway in veratridine-induced cell death of cultured bovinechromaffin cells. Nuclear condensation and fragmentation were detected several hours after a 60-min exposure to 30  A M veratridine.Apoptosis was associated with a transitory increase in p53 protein levels. Veratridine induced transcription of the pro-apoptotic p53 target gene PUMA, but not of bax or pig3. Using transient transfection experiments, we found that wild-type p53, but not the mutant form p53-273H, was sufficient to induce cell death in the chromaffin cells, which was caspase-9 dependent. The down-regulation of either p53, byoverexpressing p53-273H, or caspase-9 activity using a dominant-negative caspase-9 mutant protected chromaffin cells against veratridine-induced toxicity. Our data demonstrate the importance of p53 and the downstream activation of the mitochondrial apoptosis pathway indepolarization-induced apoptosis. D  2005 Elsevier Inc. All rights reserved. Introduction Apoptosis is an essential physiological process for theselective elimination of cells. The apoptotic pathway can be divided into three stages: a phase of induction (pre-mitochondrial), a phase of decision (mitochondrial), and a phase of degradation (post-mitochondrial). Mitochondriathat during years were postulated to be the energy supplyof eukaryotic cells have emerged as the headquarter of apoptosis signaling pathway. The decision phase, whichcontains the point of no return, corresponds to the permeabilization of the mitochondrial membranes and therelease of pro-apoptotic proteins, such as the cytochrome c , AIF, pro-caspases, and Smac/DIABLO, into the cytosol.Once in the cytoplasm Apaf-1, caspase-9 and cytochrome c  assemble and form the apoptosome, a large caspase-activating complex that plays a central role in the initiationand execution phases of apoptosis. Indeed, active forms of caspase-9 or Apaf-1 are required to form a functionalapoptosome complex, and cells expressing mutant caspase-9 are insensitive against apoptotic stimuli, includingchemotherapeutics (Osaki et al., 1997). In cellular necrosis, mitochondria also may undergo permeability transition(Ravagnan et al., 2002; Baines et al., 2005; Nakagawa et al., 2005). A large, specific pore—the mitochondrial permeability transition pore (MPTP)—opens, and solutesof up to 1.5 kDa pass freely in and out of themitochondrial matrix (Bernardi, 1999; Crompton, 1999;Jordan et al., 2003; Saelens et al., 2004).Veratridine is an alkaloid obtained from  sabadilla seeds and from the rhizome of   hellebore  that inhibitsthe complete inactivation of sodium channels, maintainingthe channel open with a small but steady sodium current  by generating a change in the three-dimensional con-formation of the sodium channels (Sutro, 1986). Vera- tridine as a depolarizing agent can also be used in the 0014-4886/$ - see front matter   D  2005 Elsevier Inc. All rights reserved.doi:10.1016/j.expneurol.2005.07.011* Corresponding author. Fax: +34 967 599 327.  E-mail address:  joaquin.jordan@uclm.es (J. Jorda´n).Experimental Neurology 196 (2005) 96 – 103www.elsevier.com/locate/yexnr   study of neuronal death, and provides a model system tostudy glutamate-independent, depolarization-induced celldeath pathways which can be relevant for white matter injury (LoPachin et al., 2001; Stys et al., 1992; Stys andLopachin, 1998). We have already shown that in bovinechromaffin cell cultures, a well-established model to studysecretory machinery (Bader et al., 2002), veratridineinduces a delayed cellular death, which has the featuresof apoptosis such as chromatin condensation and DNAfragmentation, mitochondrial de polarization, cytochrome  c release, and caspase activation (Jordan et al., 2000, 2002). Conversely, the molecular mechanisms underlying theoverall signaling response of chromaffin cells to vera-tridine toxicity are not well characterized. Wild-type p53 protein has been shown to be capable of inducingapoptosis (Yonish-Rouach et al., 1991; Ramqvist et al.,1993; Jordan et al., 1997). Recently, the roles of several p53 targets genes in mediating the p53 apoptotic responsehave been queried through loss-of-function analysis usingknockout mouse models. These studies have demonstratedthat the p53 targets including bax and PUMA (p53 up-regulated modulator of apoptosis) play cell-type-specificroles in p53-mediated apoptosis (Schuler and Green,2001). PUMA encodes a BH3-only protein and it could be a principal mediator  of cell death in response todiverse apoptotic signals (Jeffers et al., 2003).In the present study, we examined the role of p53 inveratridine-induced cell death and its implication inapoptosis of bovine chromaffin cell death. Materials and methods Chromaffin cell culture Bovine chromaffin cells were isolated as previouslydescribed (Galindo et al., 2003; Neco et al., 2004). After  washing the gland with a Ca 2+ -free Locke’s solution(Locke’s medium) containing (in mmol L  1 ): NaCl 154,KCl 5.6, MgCl 2  1, HEPES 10, glucose 10, pH 7 to removeremaining erythrocytes, adrenal glands were incubated withCa 2+ -free Locke’s medium containing 0.2% collagenase(Boehringer-Mannheim, Indianapolis, IN) and 0.5% bovineserum albumin (Calbiochem, La Jolla, CA) for 45 (3  15)min. Following medulla dissection and further incubationin collagenase solution for 30 additional minutes, chro-maffin cells were separated from erythrocytes using aPercoll gradient. Cells were plated either onto poly- l -lysine (Sigma, St. Louis, MO; 0.5 mg/mL in borate buffer,PH 8.0)-coated 15-mm round glass coverslips (2–3    10 5 cells/coverslips) for cell viability experiments or in 25 cm 2 flasks (5–8  10 6 cell/culture flasks) for reporter assays, inDulbecco’s modified Eagle’s medium (DMEM) containing10% fetal calf serum, penicillin (100 IU/mL) andstreptomycin (50  A g/mL) at 37 - C under an atmosphereof 5% CO 2 . Veratridine exposures Chromaffin cell cultures were rinsed twice with KrebsHEPES buffer (K–H) with the following ionic composition(in mM): NaCl 140, KCl 5.9, MgCl 2  1.2, HEPES 15,glucose 10, CaCl 2  2.5, pH 7.4, incubated for 1 h either inK–H or in K–H containing 30  A M veratridine at roomtemperature. Exposure was terminated by washing the cellsthree times with K–H solution. For vitamin E and cyclo-heximide treatments, drugs were added 12 h beforeveratridine exposure and maintained until the end of theexperiment. Cell viability experiments To assess the cell viability, coverslips containing chro-maffin cells were treated with 1  A g/mL Hoechst 33342 for 1min. The chromatins of GFP-positive cells stained withHoechst 33342 were examined with a standard epi-illumination fluorescence microscope (Axiophot, Zeiss,Germany). Cells with condensed or fragmented chromatinrepresented dead cells. A blinded observer counted thenumber of dead and alive cells in 10 microscopic fields(under 40  magnifications) for each coverslip and the meanwas regarded as the representative value for the coverslip.The percentage of dead cells was determined in 3 or 4coverslips for each experimental condition. The average percent apoptotic cells from at least three separate experi-ments for each condition is expressed in the text and figuresas the mean  T  SEM. Statistical significance was determined by Student’s  t   test.  Immunoblotting  Chromaffin cell cultures were washed with cold PBStwice and then collected by mechanical scraping with 1 mLof PBS per tissue culture dish. The protein suspension wascentrifuged at 12,000–14,000 rpm for 5 min. The super-natant was discarded, and the protein pellet was brought upin 150  A L of sample buffer. The protein from eachcondition was quantified spectrophotometrically (MicroBCA Protein Reagent Kit, Pierce, Rockford, IL), and anequal amount of protein (30  A g) was loaded onto each laneof the 10% SDS-PAGE, which was then run at 90 mV.After electrophoresis, proteins were transferred to Immo- bilon PVDF membranes. Non-specific protein binding was blocked with Blotto [4% w/v non-fat dried milk, 4% bovine serum albumin (Sigma) and 0.1% Tween 20(Sigma)] in PBS for 1 h. The membranes were incubatedwith anti-p53 [1:50 dilution of anti-mouse monoclonal(Pab240) sc-99 Santa Cruz] or anti-PUMA (1:1000 dilutionof polyclonal, Oncogene Research Products, San DiegoCA) overnight at 4 - C. After washing with Blotto, themembranes were incubated with a secondary antibody(1:5000 dilution of peroxidase-labeled anti-mouse, Prom-ega, Madison, WI) in Blotto. The signal was detected using  M. Gomez-Lazaro et al. / Experimental Neurology 196 (2005) 96–103  97  an enhanced chemiluminescence detection kit (AmershamECL RPN 2106 Kit). Immunoblots were developed byexposure to X-ray film (Eastman-Kodak, Rochester, NY).  Preparation of cytosolic and nuclear extracts Cells (8  10 6 ) were washed with PBS and collected bycentrifugation. Cell pellets were homogenized with 100  A Lof buffer A (10 mM HEPES [pH 7.9], 1 mM EDTA, 1 mMEGTA, 100 mM KCl, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2  A g/mL aprotinin, 10  A g/ mL leupeptin, 2  A g/mL  N  -  p -tosyl- l -lysine chloromethylketone, 5 mM NaF, 1 mM NaVO 4 , 10 mM Na 2 MoO 4 ). After 10 min at 4 - C, Nonidet P-40 was added to reach a 0.5%concentration. The tubes were gently vortexed for 15 s, andnuclei were collected by centrifugation at 8000    g   for 15min (Diaz-Guerra et al., 1996). The supernatants were stored at 80 - C (cytosolic extracts); the pellets wereresuspended in 50  A L of buffer A supplemented with 20%glycerol–0.4 M KCl and gently shaken for 30 min at 4 - C. Nuclear protein extracts were obtained by centrifugation at 13,000   g   for 15 min, and the supernatant was stored at 80 - C. Protein content was assayed using the Bio-Rad protein reagent. All cell fractionation steps were carriedout at 4 - C.  Immunostaining  Cells were fixed in 4% paraformaldehyde before permeabilization in 0.2% Triton X-100. Primary antibodyused was anti-p53 anti-mouse monoclonal (Pab240, sc-99Santa Cruz). Secondary antibody was FITC-conjugated for mouse IgG (Jackson immunoresearch). All images werecollected by a standard epi-illumination fluorescence micro-scope (Axiophot, Zeiss, Germany) and processes usingAdobe Photoshop software. Transfections For transient transfections, we used 2-day-old in vitro bovine chromaffin cells that by 24 h before transfectionswere grown in antibiotic-free media. Transfections wereachieved using Lipofectamine i  reagent (Invitrogen, Carls- bad, CA) according to the manufacturer’s protocol. Briefly,3  A L of lipofectamine reagent were pre-incubated for 20 minwith 4  A g of DNA/10 6 cells of plasmids encoding full-length, wild-type p53, p53-273H, p21 WAF-1 , retinoblastoma(provided by A. Carnero, CNIO Spain), or caspase-9dominant-negative mutant caspase-9 (C287A; casp 9DN, agift from Ding HF, Medical College of Ohio, Toledo, Ohio;Cui et al., 2002), GFP (pGFP-C1; CLONTECH Laborato-ries, Inc.), the reporter for bax, pig3, and mdm2 were a gift from X. Lu (Ludwig Institute for Cancer Research, ImperialCollege School of Medicine, London, Bergamaschi et al.,2004) and PUMA from Dr. T. Chittenden (Immunogen Inc.,Cambridge, MA). After 5 h of incubation, the transfectionmixture was removed and replaced with fresh completemedium.  Luciferase assay Luciferase activity was measured in protein extracts fromcultured bovine chromaffin cells using a Luciferase detec-tion kit (Promega, Madison, WI, USA) according to themanufacturer’s protocol. Briefly, after a 6-h treatment withveratridine (1 h, 30  A M), cells were collected andcentrifuged at 600 rpm for 10 min at room temperature,and the pellet was homogenized in 300  A L of Passive LysisBuffer. Cell homogenates were spin down, and 20  A L of  protein extracts were added to 100  A L of luciferase assay buffer containing the luciferase assay substrate and imme-diately measured in a luminometer. Luminescence wasexpressed in an arbitrary scale as relative light units (ALU). h -galactosidase ( h -gal) activity was measured according tothe manufacturer’s protocol (Promega, Madison, WI, USA)as an internal control by co-transfection of CMV- h -gal(Lecanda F, University of Navarra). Briefly, 30  A L of thesame protein extracts obtained in the Luciferase assay and20  A L of reporter lysis buffer were added to 50  A L of assay2   buffer in a 96-well plate, and incubated at 37 - C for 5 h.The reaction was stopped by adding 1 M sodium carbonateand the absorbance was measured at 415 nm in a microplatereader. Results Veratridine induces p53 protein levels Previous studies (Callaway et al., 2001; Takahashi et al.,2000) have shown that veratridine is capable of inducing celldeath in different cell types. We have previously shown that veratridine triggers DNA fragmentation and chromatincondensation in bovine chromaffin cells (Jordan et al.,2000, 2002). We have also demonstrated that veratridineinduced the generation of reactive oxygen species in bovinechromaffin cells (Jordan et al., 2000). In the first set of  experiments, we were interested in determining the contri- bution of p53 in this cell death model. Therefore, we askedwhether the total p53 protein level changed in response toveratridine by immunocytofluorescence analysis. As shownin Fig. 1A, p53 protein was undetectable in these cells under control conditions, but 6 h after veratridine treatment (30 A M, 1 h), p53-like immunoreactivity was observed in about 15% of chromaffin cells (Fig. 1B) .  Western blottingtechnique reveals that exposing cultures to veratridine (30 A M, 1 h) had a marked transitory effect on p53 protein levelsand changes in total p53 protein levels were already apparent 6 h after veratridine treatment (Fig. 1C).Since p53 is frequently found in the cytoplasm of  peripheral neurons under resting conditions and migratesto the nucleus upon activation by stress stimuli, we  M. Gomez-Lazaro et al. / Experimental Neurology 196 (2005) 96–103 98   performed subfractionation experiments to clarify the role of veratridine on p53 localization. As shown in Fig. 1D, after 6h of veratridine treatment, p53 protein was increased in thenuclear fraction. To determine whether the mechanisms of actions of veratridine on p53 depend on reactive oxygenspecies generation, we analyzed if a pre-treatment for 12 hwith the antioxidant vitamin E (50  A M) modified the levelsof p53 protein by 6 h after veratridine exposure. However,the antioxidant pre-treatment was ineffective in blocking theveratridine-induced p53 protein increase. Our results alsosuggested that p53 increases are mainly a consequence of  post-translational mechanisms, since cycloheximide (12 h,10  A g/mL) did not alter p53 protein levels after 6 h of veratridine treatment (Fig. 1E).  p53 Overexpression induces apoptosis in chromaffin cells Because p53 has been shown to promote cell death inseveral models, we tested whether p53 expression wassufficient to induce cellular death in bovine chromaffincells. Cellular cultures were co-transfected with a plasmidencoding GFP and either wild p53 (wp53) or the p53 mutant form 273 (mp53) plasmids. Morphological chromatinanalysis of GFP-positive cells by using Hoechst 33342staining demonstrated that wp53-transfected cells showedan increase in apoptosis by 48 h after transfection (Fig. 2A);and were evident morphological characteristics of apoptosisincluding cell shrink age and chromatin condensation/frag-mentation (Figs. 2B–C). In contrast, mp53-transfected cultures remained cell viability largely intact (Fig. 2A). p53 activation results in the up-regulation of different  proteins including the cyclin kinase inhibitor p21 Waf1 , whichregulate the activity of other transcription factors includingthe retinoblastoma gene product, Rb. However, overexpres-sion of  either  p21 Waf1 or Rb failed to induce significant celldeath (Fig. 2A). Fig. 1. Activation of endogenous p53 by veratridine in bovine chromaffincells. (A–B) Immunofluorescence microscopy of anti-p53 in untreatedcells (A) or cells treated with 30  A M veratridine for 1 h (B), 6 h after treatment ( n  = 3). (C–E) Whole-cell extracts (C, E) and nuclear extracts(D) from bovine chromaffin cells treated with or without veratridine (30 A M, 1 h) were subjected to Western blotting technique and probed withani-p53 antibody. Vitamin E (50  A M) and cicloheximide (CHX, 10  A g/mL)were added 12 h before veratridine and maintained until the end of theexperiment. In panels D and E, cells were collected 6 h after veratridinetreatment (30  A M; 1 h). Similar results were achieved in three independent experiments.Fig. 2. p53 overexpression induces cell death in bovine chromaffin cellcultures. (A) Bovine chromaffin cell cultures were co-transfected withexpression plasmids encoding wp53, mp53, p21 WAF1 , or retinoblastoma(Rb) together with GFP. 48 h after transfection, cells were fixed and stainedwith Hoechst 33342. GFP was used as a marker to indicate transfected cellsthat were subsequently scored for viability by chromatin morphology.Green cells were scored as either apoptotic or healthy in a blind manner.Results represent the mean  T  SEM. **  P   < 0.01 versus control conditions;ANOVA and Tukey’s test. (B–C) Pictures showing colocalization of wp53expression (B) and chromatin fragmentation measured using Hoechst 33342 (C). Arrows point to p53-transfected cells with the apoptotic phenotype. Similar results were obtained in at least 5 different experiments.  M. Gomez-Lazaro et al. / Experimental Neurology 196 (2005) 96–103  99   Effects of veratridine on p53-transcriptional activity The next set of experiments was addressed to evaluatewhether veratridine modulates some of these genes tran-scriptionally regulated by p53, in particular the pro-apoptoticgenes bax, pig3, and PUMA. Two-day-old chromaffin cellcultures were transfected with reporter construct containing p53 binding sites upstream of the luciferase gene and 24 hlater were exposed to veratridine (30  A M, 1 h). Luciferaseactivity in cell extracts was determined 6 h later. As shown inFig. 3, we detected a significant induction of luciferaseactivity in extracts from chromaffin cultures transfected withan mdm2reporter plasmid (2.13-fold increase), which servedas a positive control. Interestingly, veratridine failed toincrease the activity reporter of bax (1.13-fold increase) and pig3 (0.80-fold change).In extracts from chromaffin cultures transfected withPUMA reporter and 12 h after veratridine treatment (30  A M,1 h), we detected a significant induction of luciferase activity(1.98-fold increase). Consistently, veratridine inducedPUMA protein levels in chromaffin cells cultures (Fig. 3B). Caspase-9 knockdown blocks p53-induced cell death We have previously shown that veratridine-inducedcytochrome  c  release from mitochondria leads to caspase-3 activation in bovine chromaffin cells (Jordan et al., 2000,2002). Caspase-9 participates in the formation of theapoptosome that results in caspase-3 activation (Srinivasulaet al., 1998; Bratton et al., 2001). We therefore askedwhether the mitochondrion/caspase-9 pathway was targeted by p53 in bovine chromaffin cells. To investigate the role of caspase-9, we used the well-characterized caspase-9 dom-inant-negative mutant caspase-9 (C287A; casp9 DN) (Cui et al., 2002). Chromaffin cells were co-transfected with GFPand either  wp53 or casp9 DN or wp53 plus casp9 DN. Asshown in Fig. 4, casp9 DN expression protected chromaffin cells from the p53-induced cell death. Therefore, a func-tional caspase-9 is essential for p53-induced apoptosis in bovine chromaffin cells.  Mutant p53 overexpression blocks veratridine-induced cell death The present data point to p53 might play an important role in veratridine-induced chromaffin cell death. In order toestablish whether p53 is a key step in this death pathway,cells were transfected with mp53 plasmid and 24 h later exposed to veratridine (30  A M, 1 h). As shown in Fig. 5,mp53 overexpression affords protection in chromaffincultures against veratridine toxicity when cell viability wasassayed 24 h after treatment.Due to the fact that caspase-9 knockdown blocks p53-induced cell death in chromaffin cells, we analyzed theeffect of casp9 DN overexpression on veratridine-inducedcell death. Consistent with the idea that caspase-9 could participate in this pathway, the cultures transfected withCasp9 DN were more resistant to the veratridine exposurethan those in which the native p53 was inhibited (Fig. 5). The simultaneous inhibition of both proteins, by over-expressing mp53 and casp9 DN, did not afford a higher  protection than mp53 or casp9 DN alone (Fig. 5). Discussion We have previously shown that veratridine induces celldeath in bovine chromaffin cells throughout a mechanismthat involves an increase in mitochondrial permeabilityleading to cytochrome  c  release and caspase-3 activation(Jordan et al., 2000, 2002). In the present study, we gained insight in the apoptosis mechanisms involved in this modeland present evidence for p53 activation and the activationof caspase-9-mediated, veratridine-induced cell death.Bovine chromaffin cultures exposed to veratridineshowed the typical hallmarks of apoptosis includingchanges in birefringence, chromatin condensation, andDNA fragmentation. The tumor suppressor gene p53 wasincreased in chromaffin cell cultures exposed to veratridine(30  A M, 1 h). This increase reached a maximum 6 h after the veratridine exposure, and this was associated with anincreased nuclear localization of p53. Our results suggest that regulation of p53 by veratridine is mainly a conse- Fig. 3. Effects of veratridine treatments on p53-transcriptional activity. (A)Two-day-old chromaffin cell cultures were transfected with reporter construct containing p53 binding sites upstream of the luciferase geneand 24 h later were exposed to veratridine (30  A M, 1h). Luciferase activityin cell extracts was determined 6 h later. Luminescence was expressed in anarbitrary scale as relative lights units (ALU).  h -galactosidase ( h -gal)activity was measured as an internal control. Values are the mean  T  SD of four experiments. **  P   < 0.01 versus control conditions; ANOVA andTukey’s test. (B) Immunoblot analysis of PUMA protein levels in whole-cell extracts from control and veratridine-treated chromaffin cells at theindicated times points after exposures (30  A M, 1 h). Similar results werefound in three separate experiments.  M. Gomez-Lazaro et al. / Experimental Neurology 196 (2005) 96–103 100
Related Documents
View more...
We Need Your Support
Thank you for visiting our website and your interest in our free products and services. We are nonprofit website to share and download documents. To the running of this website, we need your help to support us.

Thanks to everyone for your continued support.

No, Thanks
SAVE OUR EARTH

We need your sign to support Project to invent "SMART AND CONTROLLABLE REFLECTIVE BALLOONS" to cover the Sun and Save Our Earth.

More details...

Sign Now!

We are very appreciated for your Prompt Action!

x