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INTEGRATIVE |
1 Center for Neuroscience
4 Institute of Biomedical Science, National Sun Yat-sen University, Kaohsiung, Taiwan, Republic of China
2 Center for Gene Regulation and Signal Transduction Research, National Cheng Kung University, Tainan, Taiwan, Republic of China
3 Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan, Republic of China
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Abstract |
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(Received 3 April 2006;
accepted after revision 28 April 2006;
first published online 4 May 2006)
Corresponding author S. H. H. Chan: Center for Neuroscience, National Sun Yat-sen University, Kaohsiung 80424, Taiwan, Republic of China. Email: schan{at}mail.nsysu.edu.tw
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Introduction |
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Our laboratory demonstrated previously that the RVLM plays a pivotal role in the manifestation of fatal circulatory depression in an experimental endotoxaemia model of brain stem death. We showed that the repertoire of cellular events that underlie this cardiovascular fatality entails mitochondrial dysfunction (Chuang et al. 2002, 2003; Chan et al. 2005d) induced by an excessive production of nitric oxide (NO) generated by NO synthase II (NOS II) (Chan et al. 2001, 2002, 2004b, 2005d; Li et al. 2005), superoxide anion (Chuang et al. 2003; Chan et al. 2005d) or peroxynitrite (Chan et al. 2002, 2005d; Li et al. 2005), followed by the release of cytochrome c to the cytosol that activates caspase-9 and caspase-3, leading to apoptotic cell death in the RVLM (Chan et al. 2005d).
To counteract those ‘pro-death’ programmes, we proposed that ‘pro-life’ programmes in the RVLM must also be activated during the progression towards brain stem death (Chan et al. 2005b). Likely candidates for those ‘pro-life’ programme include the heat-shock proteins (HSPs). The HSPs represent a group of intracellular proteins that are highly conserved across species and are thought to participate in protective adaptation that spares cells from otherwise lethal consequences of exposure to elevation in temperature, toxic conditions, infection or cellular stress (Lindquist & Craig, 1988). As molecular chaperones (Ellis & van der Vries, 1991), HSPs confer neuroprotection by their critical role in intracellular processing, synthesis, transportation and degradation of proteins. Ample evidence also links HSP70 and HSP90 to protection against apoptosis (Arrigo, 1998; Jolly & Morimoto, 2000; Xanthoudakis & Nicholson, 2000).
In eukaryotic cells, the majority of 60 kDa HSPs are localized to the mitochondria (Soltys & Gupta, 1996), with minor presence in the cytosol (Kirchhoff et al. 2002). Whereas the mitochondrion plays an important role in the pathogenesis of endotoxaemia by initiating apoptotic cell death (Fink, 2001; Boveris et al. 2002; Brealey et al. 2004; Chan et al. 2005d), controversies exist regarding the role of HSP60 in apoptosis. HSP60 is reportedly pro-apoptotic in Jurkat cells (Samali et al. 1999) but anti-apoptotic in cardiac myocytes (Lin et al. 2001; Kirchhoff et al. 2002; Shan et al. 2003). The present study assessed the hypothesis that HSP60 reduces cardiovascular fatality in an experimental endotoxaemia model of brain stem death by an anti-apoptotic action in the RVLM. Our combined biochemical, physiological and pharmacological results validated this hypothesis. We further showed that HSP60 redistributed from mitochondrion to cytosol reduces the activation of the cytochrome c–caspase-3 cascade of apoptotic signalling via enhanced interactions with the mitochondrial or cytosolic Bax or Bcl-2.
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Methods |
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General preparation
As in our previous studies (Chan et al. 2001, 2002, 2004b, 2005a,c; Chuang et al. 2002, 2003; Li et al. 2005), preparatory surgery that included cannulation of femoral artery or both femoral veins and tracheal intubation was performed under an induction dose of pentobarbital sodium (50 mg kg–1, I.P.). During the recording session, which routinely commenced 60 min after the administration of pentobarbital sodium, anaesthesia was maintained by an intravenous infusion of propofol (Zeneca, Macclesfield, UK) at 20–25 mg kg–1 h–1. This scheme provided satisfactory anaesthetic maintenance while preserving the capacity of central cardiovascular regulation (Yang et al. 1995). During the recording session, the body temperature of the animals was maintained at 37°C with a heating pad, and animals were allowed to breathe spontaneously with room air via the intubated trachea.
Recording and power spectral analysis of systemic arterial pressure (SAP) signals
SAP signals recorded from the femoral artery were simultaneously subject to on-line power spectral analysis (Chan et al. 2001, 2002, 2004b, 2005a,c; Chuang et al. 2002, 2003; Li et al. 2005). We were particularly interested in the low-frequency (LF; 0.25–0.8 Hz) and very low-frequency (VLF; 0–0.25 Hz) components in the SAP spectrum. Our laboratory demonstrated previously that those spectral components of SAP signals take origin from the RVLM (Kuo et al. 1997a), and their power density reflects the prevailing sympathetic neurogenic vasomotor tone (Chan et al. 2001, 2005a; Li et al. 2005), as well as the functional integrity of the brain stem (Kuo et al. 1997a,b; Yien et al. 1997; Yen et al. 2000). Heart rate (HR) was derived instantaneously from SAP signals. The SAP spectra and power density of the two spectral components were displayed during the experiment, alongside pulsatile SAP, mean SAP (MSAP) and HR, in an on-line and real-time manner.
Experimental endotoxaemia model of brain stem death
An experimental endotoxaemia model (Chan et al. 2005b), which mimics clinically the progression towards brain stem death during systemic inflammatory response syndrome (Yien et al. 1997), was used in the present study. In essence, Escherichia coli lipopolysaccharide (LPS; serotype 0111:B4; Sigma-Aldrich, St Louis, MO, USA) was administrated intravenously over 1–2 min at 30 mg kg–1 (Chan et al. 2001; Chuang et al. 2002, 2003; Li et al. 2005). Temporal changes in MSAP, HR or power density of LF or VLF component of the SAP signals were routinely followed for 240 min, or until the animal succumbed to endotoxaemia. The survival rate within 240 min was also recorded. Intravenous administration of saline served as the vehicle control.
Collection of tissue samples from ventrolateral medulla
As we reported previously (Chan et al. 2001, 2004b, 2005d; Chuang et al. 2002, 2003; Li et al. 2005), the sequence of cardiovascular events during LPS-induced endotoxaemia can be divided into three phases. At the peak of each of these phases of experimental endotoxaemia (LPS group), or 30, 150 or 240 min after intravenous injection of saline (saline group), animals were killed with an overdose of pentobarbital sodium, and tissues on both sides of the ventrolateral part of medulla oblongata, at the level of RVLM (0.5–2.5 mm rostral to the obex), were collected and processed for subsequent biochemical evaluations. Tissues obtained from animals that were anaesthetized and received preparatory surgery served as our sham-controls. Protein concentration was determined by the BCA Protein Assay (Pierce, Rockford, IL, USA).
Proteomic analysis
Proteomic analysis of proteins extracted from the ventrolateral medulla was carried out as described previously (Huang et al. 2002; Li et al. 2005). The silver-stained 2-dimensional electrophoresis gels in the domain of isoelectric point (pI) 3–10 and molecular mass 14.4–94 kDa were scanned in an ImageScanner (Amersham Pharmacia Biotech, Uppsala, Sweden). Protein spots were quantified and numbered using the ImageMaster 2D Elite software (Amersham Pharmacia Biotech). The protein level of each spot was expressed as a percentage of total spot volume in the 2-dimensional electrophoresis gel. Protein spots within the domain of pI 3–10 and mol. mass 43–67 kDa in the Coomassie brilliant blue-stained 2-dimensional electrophoresis gels were further subject to in-gel digestion, and analysed by MALDI-TOF mass spectrometry (Voyager ED-PRO, Applied Biosystems, Foster City, CA, USA). To identify proteins, the measured mono-isotopic masses of peptide were routinely analysed using MS-Fit (Protein Prospector, UCSF Mass Spectrometry Facility, San Francisco, CA, USA) and MASCOT (Matrix Science, Boston, MA, USA) search programs.
Western blot analysis
Western blot analysis (Chan et al. 2002, 2004b, 2005a,c; Li et al. 2005) was carried out on proteins extracted from the ventrolateral medulla for HSP60, Bax, Bcl-2, cytochrome c, activated caspase-3, prohibitin or ß-actin. The primary antisera used included goat polyclonal antiserum against HSP60 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); rabbit polyclonal antiserum against Bax (Cell Signaling, Beverly, MA, USA) or activated caspase-3 (Cell Signaling); or mouse monoclonal antiserum against Bcl-2 (Santa Cruz), cytochrome c (Santa Cruz), prohibitin (Labvision/NeoMarkers, Fremont, CA, USA) or ß-actin (Chemicon, Temecula, CA, USA). The secondary antisera used included horseradish peroxidase-conjugated donkey anti-goat IgG (Santa Cruz) for HSP60; donkey anti-rabbit IgG (Amersham Biosciences, Little Chalfont, UK) for Bax or activated caspase-3; or sheep anti-mouse IgG (Amersham Biosciences) for Bcl-2, cytochrome c, prohibitin or ß-actin. Specific antibody–antigen complex was detected by an enhanced chemiluminescence Western blot detection system (NEN, Boston, MA, USA). The amount of protein was quantified by the ImageMaster software (Amersham Pharmacia Biotech), and was expressed as a ratio relative to ß-actin protein (for analysis of total protein or proteins in cytosolic fraction) or prohibitin (for analysis of proteins in mitochondrial fraction).
Isolation of RNA and real-time polymerase chain reaction (PCR)
Total RNA from the ventrolateral part of medulla oblongata was isolated with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) (Chan et al. 2004b, 2005c). All RNA isolated was quantified by spectrophotometry and the optical density (OD) 260/280 nm ratio was determined. As in our recent study (Chan et al. 2005c), reverse transcriptase reaction was performed using a SuperScript Preamplification System (Invitrogen) for the first-strand cDNA synthesis. Real-time PCR analysis was performed by amplification of cDNA using a LightCycler instrument (Roche Diagnostics, Mannheim, Germany). PCR reaction for each sample was carried out in duplicate for all the cDNA and for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control. The PCR mixture (total volume, 20 µl), which was prepared with nuclease-free water, contained 2 µl of LightCycler FastStart DNA Master SYBR Green 1 (Roche Diagnostics), 3 mM MgCl2 and 5 µM of each primer, together with 5 µl of purified DNA or negative control. Primers for hsp60 and GAPDH were designed using the Roche LightCycler probe design software 2.0 and based on sequence information of the NCBI database, and oligonucleotides were synthesized by Genemed Biotechnologies (Taipei, Taiwan). The primer pairs for amplification of hsp60 cDNA (GenBank accession no. NM022229) were 5'-AGGCATGAAGTTTGATAGAGG-3' for the forward primer, and 5'-TTFFCAATTTCAAGAGCAGG-3' for the reverse. Primer pairs for GAPDH cDNA (GenBank accession no. NM017008) were 5'-GCCAAAAGGGTCATCATCTC-3' for the forward primer, and 5'-GGCCATCCACAGTCTTCT-3' for the reverse. The amplification protocol for cDNA on the LightCycler was a 10 min denaturation step at 95°C for polymerase activation, a ‘touch down’ PCR step of 10 cycles consisting of 10 s at 95°C, 10 s at 65°C, and 30 s at 72°C, followed by 40 cycles consisting of 10 s at 95°C, 10 s at 55°C, and 30 s at 72°C. After slow heating (0.1°C s–1) of the amplified product from 65 to 95°C to generate a melting temperature curve, which serves as a specificity control, the PCR samples were cooled to 40°C (Emrich et al. 2002). Fluorescence signals from the amplified products were quantitatively assessed using the LightCycler software program (version 3.5). Second derivative maximum mode was chosen with baseline adjustment set in the arithmetic mode. The relative change in hsp60 mRNA expression was determined by fold-change analysis (Winer et al. 1999; Chan et al. 2005c) in which Fold change = 2–[
Ct], where Ct = (Cthsp60
– CtGAPDH)LPS treatment
– (Cthsp60
– CtGAPDH)sham control. Note that the Ct value is the cycle number at which the fluorescence signal crosses the threshold.
Microinjection of test agents
The bilateral microinjection of test agents into the RVLM, each at a volume of 50 nl, was carried out stereotaxically and sequentially. The coordinates used were: 4.5–5 mm posterior to lambda, 1.8–2.1 mm lateral to midline, and 8.1–8.4 mm below the dorsal surface of the cerebellum (Chan et al. 2001, 2002, 2004b, 2005a,c; Chuang et al. 2002, 2003; Li et al. 2005). Test agents used included: a transcription inhibitor (Fernando et al. 2000; Li et al. 2005), actinomycin D (Tocris Cookson, Bristol, UK); a translation inhibitor (Fernando et al. 2000; Li et al. 2005), cycloheximide (Tocris Cookson); normal goat serum (Sigma-Aldrich), goat polyclonal antiserum against HSP60 (Santa Cruz); or sense (5'-GATGCTCGAGCCTTA-3'), antisense (5'-TAAGGCTCGAGCATC-3') or scrambled (5'-GCTCGTGGTCAATAC-3') oligonucleotide (Quality Synthesis, Taipei, Taiwan) against hsp60 (Kirchhoff et al. 2002). The dose and treatment regimen were adopted from previous reports (Fernando et al. 2000; Kirchhoff et al. 2002; Li et al. 2005) that used those test agents for the same purpose as in this study. Actinomycin D was prepared with 0.1% DMSO, and cycloheximide with 50% ethanol (EtOH). We added 0.02% Triton X-100 (Sigma-Aldrich) to anti-HSP60 antiserum to facilitate its transport across the cell membrane (Li et al. 2005). All oligonucleotides were phosphorothioated in all positions and were diluted in artificial cerebrospinal fluid (aCSF) at pH 7.4. The composition of aCSF was (mM): NaCl 117, NaHCO3 25, glucose 11, KCl 4.7, CaCl2 2.5, MgCl2 1.2 and NaH2PO4 1.2. Microinjection of 0.1% DMSO, 50% EtOH, normal goat serum plus Triton X-100 or aCSF served as our vehicle and volume control. To avoid the confounding effects of drug interactions, each animal received only one pharmacological treatment.
Qualitative and quantitative analysis of DNA fragmentation
Total DNA was extracted from samples of the ventrolateral medulla and nucleosomal DNA ladders were separated by electrophoresis on a 1% agarose gel (Chan et al. 2005d), after amplification using a PCR kit for DNA ladder assay (APO-DNA1, Maxim Biotech, South San Francisco, CA, USA) to enhance the detection sensitivity. To quantify apoptosis-related DNA fragmentation, a cell death enzyme-linked immunosorbent assay (Roche Molecular Biochemicals, Mannheim, Germany) that detects apoptotic but not necrotic cell death (Bonfoco et al. 1995) was used to assay the level of histone-associated DNA fragments in the cytoplasm (Wu et al. 2003; Saito et al. 2004). In brief, proteins from the cytosolic fraction of the ventrolateral medullary samples isolated by a nuclear extraction kit (Active Motif, Carlsbad, CA, USA) were used as the antigen source, together with primary anti-histone antiserum or secondary anti-DNA antiserum coupled to peroxidase. The amount of nucleosomes in cytoplasm was quantitatively determined using 2,2'-azino-di-[3-ethylbenzthiazoline sulphonate] as the substrate. Absorbance was measured at 405 nm and referenced at 490 nm using an ELISA microtitre plate reader (Anthros Labtec, Salzburg, Austria).
Double immunofluorescence staining and laser confocal microscopy
Rats were perfused transcardially with warm isotonic saline solution, followed by ice-cold 3.8% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.2). The brain stem was removed, postfixed by submersion in the latter solution overnight at 4°C and cryoprotected by 30% sucrose in 0.1 M PBS. Frozen transverse sections (30 µm) of the medulla oblongata were cut on a cryostat (Leitz Cryostat 1729, Welzlar, Germany) and collected in 0.1 M PBS. The double immunofluorescence staining procedures were modified from those reported previously (Chan et al. 2004a). In brief, free-floating sections of the medulla oblongata were incubated with a rabbit polyclonal antiserum against activated caspase-3 (Cell Signaling), together with a mouse monoclonal antiserum directed against a specific neuron marker (Mullen et al. 1992), neuron-specific nuclear protein (NeuN; Chemicon). The sections were subsequently incubated concurrently with two appropriate secondary antisera (Molecular Probes, Eugene, OR, USA), including a goat anti-rabbit IgG conjugated with Alexa Fluor 488 for activated caspase-3 and a goat anti-mouse IgG conjugated with Alexa Fluor 568 for NeuN. Viewed under a Fluorview FV300 laser scanning confocal microscope (Olympus, Tokyo, Japan), immunoreactivity for NeuN exhibited red fluorescence and activated caspase-3 exhibited green fluorescence. The occurrence of yellow fluorescence or co-localization of red and green fluorescence on merged images indicated the presence of activated caspase-3 immunoreactivity in neurons.
Isolation of cytosolic and mitochondrial fractions
A mitochondrial extraction kit (Active Motif) was used to isolate the cytosolic and mitochondrial fractions of samples from the ventrolateral medulla. In essence, tissue samples were gently homogenized with a glass–glass homogenizer. Homogenates were centrifuged at 800 g for 10 min at 4°C, and the supernatant was collected and centrifuged at 16 000 g for another 30 min at 4°C to pellet the mitochondria. The supernatant thus obtained was the cytosolic fraction. The mitochondrial pellet was resuspended in 100 µl of isolation medium. The purity of the mitochondrial fraction was verified by the selective expression of the mitochondrial inner membrane-specific proteins prohibitin or manganese superoxide dismutase (Stressgen, Victoria, Canada), or copper-zinc superoxide dismutase (Stressgen) in the cytosolic fraction (Chan et al. 2005d). Total protein in the mitochondrial or cytosolic extracts was estimated by the BCA Protein Assay (Pierce).
Immunoprecipitation and immunoblot analysis
Protein extracts from either the mitochondrial or cytosolic fraction of samples from the ventrolateral medulla were immunoprecipitated with affinity-purified goat polyclonal anti-HSP60, rabbit polyclonal anti-Bax or mouse monoclonal anti-Bcl-2 antiserum conjugated with protein G–agarose beads (Santa Cruz). Immunoprecipitation was performed at 4°C overnight and the precipitated beads obtained after being centrifuged for 5 s at 6000 g. were washed three times with ice-cold lysis buffer. The agarose beads resuspended in the loading buffer were boiled for 5 min to dissociate the immunocomplexes from the beads. Western blot analysis of Bax, Bcl-2 or HSP60 from proteins immunoprecipitated by anti-HSP60 antiserum, HSP60 or Bax from proteins immunoprecipitated by anti-Bax antiserum, or HSP60 or Bcl-2 from proteins immunoprecipitated by anti-Bcl-2 antiserum was carried out as described above.
Histology
In some experiments, the brain stem was removed after the physiological experiment and fixed in 10% formaldehyde in 30% sucrose solution for at least 72 h. Histological verification of the microinjection site was performed on 25 µm frozen sections stained with Neutral Red.
Statistical analysis
All values are expressed as mean ± S.E.M. The averaged value of MSAP or HR calculated every 20 min after administration of test agents or vehicle, the sum total of power density for the LF or VLF component in the SAP spectra over 20 min, or the protein expression level or the level of histone-associated DNA fragments in the ventrolateral medulla during each phase of experimental endotoxaemia were used for statistical analysis. One-way or two-way analysis of variance with repeated measures was used, as appropriate, to assess group means, followed by Scheffé's multiple-range test for post hoc assessment of individual means. Mortality rate was assessed by the Fisher exact test. P < 0.05 was considered statistically significant.
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Results |
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Our first series of experiments established the presence of HSP60 in the RVLM by subjecting total proteins extracted from the ventrolateral medulla of adult Sprague-Dawley rats to proteomic analysis. Proteomic maps (Fig. 1A) generated for the ventrolateral medulla, coupled with protein finger-printing based on MALDI-TOF mass spectrometry (Fig. 1B) and complementary search results using both MS-Fit and MASCOT programs (Huang et al. 2002; Li et al. 2005), consistently revealed the presence of HSP60 (SWISS-PROT accession no. P19226) in the RVLM. The relative contribution of HSP60 to the total spot volume in the proteomic map (pI, 3–10; mol. mass, 14.4–94 kDa) of the RVLM was 0.16 ± 0.02% (mean ± S.E.M. of duplicate analyses on samples from 6 different animals).
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As reported previously (Chan et al. 2001, 2004b, 2005d; Chuang et al. 2002, 2003; Li et al. 2005), based on the decrease, increase, and a secondary decrease in the power density of the LF or VLF component in the SAP spectrum, the sequence of cardiovascular responses induced by intravenous administration of LPS (30 mg kg–1) can be divided into three phases (cf. Fig. 4). Both proteomic (Fig. 1C–G) and Western blot (Fig. 1H) analyses in our second series of experiments revealed that HSP60 protein in total homogenates of the ventrolateral medulla manifested a progressive elevation in expression that became significant during phases II and III of endotoxaemia. The level of HSP60 in the RVLM of saline-control rats was stable, and was comparable to sham-controls.
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Whether the augmented HSP60 expression in the ventrolateral medulla during experimental endotoxaemia entailed de novo synthesis was investigated in our third series of experiments. Real-time PCR analysis revealed that hsp60 mRNA underwent a progressive and significant increase during phases II and III of endotoxaemia (Fig. 2A). Western blot analysis further showed that the progressive elevation in HSP60 protein expression was significantly blunted (Fig. 2B) in animals that were pretreated with microinjection of a transcription inhibitor, actinomycin D (5 nmol) into the bilateral RVLM, 1 h prior to intravenous administration of LPS (30 mg kg–1). Animals that were similarly pretreated with a translation inhibitor, cycloheximide (20 nmol) succumbed to endotoxaemia within 10 min of LPS administration, without manifesting the phasic cardiovascular responses or the surge in HSP60 expression in the ventrolateral medulla (Fig. 2B). Pretreatment with either vehicle (0.1% DMSO or 50% EtOH) did not affect HSP60 level in saline-control animals or prevent the augmentation in HSP60 level in the ventrolateral medulla during phases II and III of endotoxaemia (Fig. 2B).
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Our fourth series of experiments employed two loss-of-function manipulations (immuno-neutralization and gene knockdown) to establish a causative relationship between HSP60 expression, survival and cardiovascular protection during experimental endotoxaemia. Given over 1 min at the intravenous dose (30 mg kg–1) that we used, LPS elicited approximately 60% fatality within 240 min of administration (Fig. 3A). Pretreatment with microinjection into the bilateral RVLM of an anti-HSP60 antiserum (1 : 20) 1 h before the induction of experimental endotoxaemia resulted in 100% mortality by 180 min after injection of LPS (Fig. 3A). Similarly, all animals pretreated with local application of an antisense hsp60 oligonucleotide (50 pmol) into the bilateral RVLM 24 h before administration of LPS died 190–200 min after the induction of endotoxaemia (Fig. 3A); this was coincidental to an antagonism of the augmented HSP60 expression in the ventrolateral medulla during phases II and III of endotoxaemia (Fig. 3B).
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The augmented HSP60 expression in ventrolateral medulla during experimental endotoxaemia exerts an anti-apoptotic action
In animals that received pretreatment with a microinjection of normal goat serum (1 : 20) into the bilateral RVLM, apoptosis with characteristic nucleosomal DNA ladder (Fig. 5, upper panel) or cytoplasmic histone-associated DNA fragments (Fig. 5, lower panel) was detected in the ventrolateral medulla during experimental endotoxaemia, with progressively heightened intensity (Chan et al. 2005d). This manifested apoptotic cell death, while absent in saline-control animals, was significantly enhanced in animals pretreated with an anti-HSP60 antiserum (Fig. 5).
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Two approaches were employed in our sixth series of experiments to identify the intracellular mechanisms that may underlie the anti-apoptotic action of HSP60 in the RVLM during experimental endotoxaemia. Based on immuno-neutralization, we first evaluated whether translocation of Bax, Bcl-2, activated caspase-3 or cytochrome c is causatively related to HSP60 expression in the ventrolateral medulla during experimental endotoxaemia. In rats pretreated with normal goat serum (1 : 20), Western blot analysis revealed that the progressive elevation of HSP60 level in total homogenates observed during experimental endotoxaemia (Figs. 1 and 2) actually involved a gradual decrease in the mitochondrial fraction of samples from the ventrolateral medulla, alongside a progressive increase in the cytosolic fraction (Fig. 7). The level of Bcl-2 or cytochromec, which was primarily localized in the mitochondrial fraction, underwent a progressive decrease during experimental endotoxaemia, along with a gradual increase in Bax (Fig. 7). Correspondingly, there was a progressive augmentation in HSP60, Bcl-2, cytochrome c or activated caspase-3, together with a gradual reduction in Bax, in the cytosolic fraction (Fig. 7). Intriguingly, whereas those manifested features of cytochrome c-dependent apoptotic mechanism in the ventrolateral medulla during experimental endotoxaemia (Chan et al. 2005d) were absent in saline-control animals, they were exacerbated in animals pretreated with a microinjection of an anti-HSP60 antiserum into the bilateral RVLM (Fig. 7).
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Discussion |
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Intracellular redistribution of the augmented HSP60 in the RVLM during experimental endotoxaemia
Complementary data from our proteomic and Western Blot analyses revealed that HSP60 manifested a progressive augmentation in its expression level during phases II and III of endotoxaemia. Our observations with real-time PCR analysis and Western blot analysis coupled with actinomycin D or cycloheximide pretreatment further showed that this elevated HSP60 was the result of de novo synthesis (Oksala et al. 2004). Being a nuclear-coded protein (Picketts et al. 1989; Reading et al. 1989) with heat shock element present in the promotor region (Ryan et al. 1997), it is conceivable that the hsp60 gene is subject to the regulation of heat shock transcription factor 1 (Morimoto, 1998). Accumulation of unfolded proteins within the mitochondrial matrix may also result in selective transcriptional activation of the mitochondrial stress response element in the hsp60 gene (Zhao et al. 2002). Intriguingly, superimposed on the augmented expression was a progressive decline in mitochondrial or elevation in cytosolic HSP60 in the ventrolateral medulla. We reason that this redistribution of HSP60 may arise from at least two, albeit not mutually exclusive, sources. First, it is conceivable that the newly synthesized HSP60 has a higher cytosolic presence. Second, the redistribution of HSP60 is the result of mitochondrial to cytosolic translocation (Samali et al. 1999).
The redistributed HSP60 in the RVLM is neuroprotective and anti-apoptotic
Extra-mitochondrial expression of HSP60 has been associated with cellular responses to inflammatory stress. For example, autologous HSP60 induces LPS tolerance by reducing the responsiveness of hepatic Kupffer cells to a subsequent LPS challenge (Schuchmann et al. 1999). Cell surface expression of HSP60 is recognized as a danger signal to the innate immune system, which stimulates macrophages to express IL-12 and IL-15 or releases TNF-
(Chen et al. 1999). In this regard, a defined region of HSP60 is involved in LPS binding, which mediates its macrophage stimulatory effects (Habich et al. 2005). Whether these mechanisms also take place in the RVLM during experimental endotoxaemia remain to be evaluated.
Samali et al. (1999) demonstrated in Jurkat cells that the induction of apoptosis involves mitochondrial to cytosolic translocation of HSP60 or cytochrome c, concomitant with activation of mitochondrial procaspase-3. In addition, the released HSP60 may participate in amplification of the pro-apoptotic cytochrome c–caspase-3 cascade. Our observations that the progressive decrease in mitochondrial, or gradual increase in cytosolic, HSP60 in the ventrolateral medulla during experimental endotoxaemia was accompanied by a parallel elevation in nucleosomal DNA fragmentation or cytoplasmic histone-associated DNA fragments, or augmentation in cytosolic cytochromec or activated caspase-3 level, are therefore of interest. One plausible interpretation is that, as a cellular response to inflammatory stress, the redistributed HSP60 induces apoptotic cell death in the RVLM, in concert with the activated cytochrome c–caspase-3 signalling cascade. Complementary observations from the present study argue strongly against this interpretation, and support instead the notion that HSP60 in the RVLM confers neuroprotection against fatal cardiovascular depression during endotoxaemia via an anti-apoptotic action. By showing that loss-of-function manipulations of HSP60 exacerbated mortality and potentiated cardiovascular depression induced by LPS, we established a causative relationship between the augmented HSP60 expression in the ventrolateral medulla and survival from, or cardiovascular protection against, fatal endotoxaemia. The fact that functional blockade of HSP60 in the RVLM by an anti-HSP60 antiserum intensified the experimental apoptotic indices or augmented the cytochromec–caspase-3 cascade of apoptotic signalling in the RVLM further substantiated our notion. In this regard, HSP60 has also been proposed to play an anti-apoptotic role in cardiac myocytes (Lin et al. 2001; Kirchhoff et al. 2002; Shan et al. 2003).
Novel anti-apoptotic mechanisms of HSP60 involve enhanced interactions with Bax or Bcl-2
One of the decisive steps of the apoptotic cascade is permeabilization of the outer mitochondrial membrane (Kroemer, 1999; Crompton, 2000), which leads to the release of cytochrome c from the intermediate space, followed by the activation of caspase-dependent cascade of apoptotic signalling. It is generally contended (Kroemer, 1999; Lossi & Merighi, 2003) that whereas the anti-apoptotic members of the Bcl-2 family (e.g. Bcl-2) work to prevent cytochromec release by stabilizing the mitochondrial membrane barrier function (Yang et al. 1997), the pro-apoptotic members (e.g. Bax) tend to induce cytochrome c release by permeabilizing the mitochondrial membrane (Jürgensmeier et al. 1998). Translocation of Bax from cytosol to mitochondrion is induced during apoptosis, and this process is inhibited by Bcl-2 (Kirchhoff et al. 2002; Hou & Hsu, 2005). Our observed progressive translocation of cytosolic Bax to the mitochondria, alongside a gradual shift of mitochondrial Bcl-2 to the cytosol and an increased cytosolic presence of cytochrome c or activated caspase-3, are indicative of an interplay between Bax or Bcl-2 and cytochrome c-dependent apoptotic cell death in the RVLM during experimental endotoxaemia. More importantly, we provided novel results to suggest that the redistributed HSP60 in the RVLM exerts its anti-apoptotic action during experimental endotoxaemia by interacting with this interplay between Bax and Bcl-2.
In cardiac myocytes, the cytosolic HSP60 plays an anti-apoptotic role by complexing with Bax (Kirchhoff et al. 2002; Shan et al. 2003). It is therefore intriguing to note that our immunoprecipitation and immunoblot analysis revealed that the progressive mitochondrial to cytosolic redistribution of HSP60 in the RVLM during experimental endotoxaemia was accompanied by a gradual augmentation of the complex formed between HSP60 and Bax in the cytosolic fraction. These findings indicate that, by increasing its interaction with Bax, the enhanced presence of HSP60 in the cytosol functions to prevent the translocation of this pro-apoptotic factor to the mitochondria (Kirchhoff et al. 2002; Hou & Hsu, 2005). A novel finding in this study is that HSP60 also complexed with Bax in the mitochondrial fraction under basal conditions, and this complex was progressively enlarged during endotoxaemia. Such an intensified interaction between HSP60 and Bax in the mitochondria may conceivably prevent the oligomerization and insertion of Bax into the mitochondrial membrane, two essential steps for triggering cytochrome c release (Lossi & Merighi, 2003). The finding that pretreatment with an anti-HSP60 antiserum augmented the mitochondrial, but reduced the cytosolic, presence of Bax in the RVLM during experimental endotoxaemia provided ample credence to these notions.
Two additional novel observations on Bcl-2 were unveiled in the present study. First, we demonstrated that HSP60 also complexed with both mitochondrial and cytosolic Bcl-2 in the ventrolateral medulla. Second, there is a gradual shift of mitochondrial Bcl-2 to the cytosol during experimental endotoxaemia in the RVLM. More importantly, this redistribution of Bcl-2 is not simply a passive consequence of the progressive mitochondrial to cytosolic shift of HSP60. By progressively enhancing its interaction with Bcl-2 in the mitochondria, the gradually reduced mitochondrial HSP60 may help prevent apoptotic cell death by strengthening the capability of Bcl-2 to retard the release of cytochrome c to the cytosol. On the other hand, Bcl-2 dissociated from HSP60 in the cytosol may reinforce its anti-apoptotic action by preventing the translocation of Bax to the mitochondria (Kirchhoff et al. 2002; Hou & Hsu, 2005). Again, the fact that pretreatment with an anti-HSP60 antiserum further decreased the mitochondrial, but increased the cytosolic, level of Bcl-2 in the RVLM during endotoxaemia provided ample support for these notions.
Biochemical (Gupta & Austin, 1987) and morphological (Soltys & Gupta, 1996) studies revealed that HSP60 is primarily present in the matrix or inner membrane. On the other hand, it is generally contended (Kroemer, 1999; Lossi & Merighi, 2003) that Bcl-2 resides on the mitochondrial membrane, and Bax is either cytosolic or present on the cytoplasmic surface of the outer mitochondrial membrane. As such, a seeming discrepancy exists between our stipulation that HSP60 plays a crucial anti-apoptotic role by interacting with Bax and Bcl-2 in the RVLM during experimental endotoxaemia and the difference in their localization in the mitochondrial compartments. This discrepancy is minimized by our observations that whereas Bax or Bcl-2 precipitated large amounts of mitochondrial HSP60, only a fraction of the precipitated HSP60 bound with Bax or Bcl-2. These findings confirmed the fact that while most of the Bax or Bcl-2 in the mitochondria are associated with HSP60, only a portion of the 60 kDa HSP forms a complex with these two Bcl-2 family members. At the same time, they are in line with the report (Soltys & Gupta, 1996) that whereas HSP60 is present primarily in the matrix compartment, it also exists on the cytoplasmic side of the mitochondrial outer membrane. We also verified the purity of the mitochondrial fraction by the selective expression of the mitochondrial inner membrane-specific proteins prohibitin or manganese superoxide dismutase, and copper-zinc superoxide dismutase in the cytosolic fraction (Chan et al. 2005d).
Anti-apoptotic actions of HSP60 may also involve restoration of mitochondrial functions
Overexpression of HSP60 results in protection of cardiac myocytes against simulated ischaemic and reoxygenation-induced apoptotic cell death (Lin et al. 2001). The underlying mechanisms for this anti-apoptotic action of HSP60 include preservation of oxidative phosphorylation, accelerated ATP recovery, decreased cytochrome c release or caspase-3 activation. Excessive NO produced during sepsis affects oxidative phosphorylation by inhibiting the mitochondrial respiratory enzymes (Podcroso et al. 1996), and the resultant mitochondrial dysfunction induces apoptosis (Carreras et al. 2004). Generation of reactive oxygen species, in particular superoxide anion, because of a deficiency in the mitochondrial electron transport chain (Podcroso et al. 1996; Carreras et al. 2004) mediates apoptosis in peripheral tissues during sepsis (Li et al. 2002). Mitochondrial dysfunction also triggers downstream caspase cascades via the release of pro-apoptotic factors, including cytochrome c, to the cytosol (Kroemer, 1999). We demonstrated recently (Chan et al. 2005d) that, in the RVLM, LPS promotes NO- and superoxide anion-dependent depression of the mitochondrial respiratory enzyme Complex I and IV activities and subsequent release of cytochrome c from the mitochondria to the cytosol. This was followed by activation of caspase-9 and caspase-3, leading to apoptotic cell death in the RVLM. It is noteworthy that microinjection of the electron carrier coenzyme Q10 into the RVLM reverses the manifested mitochondrial dysfunction and induced apoptotic cell death. It follows that the augmented HSP60 in the RVLM may confer cardiovascular protection via the same anti-apoptotic mechanisms observed in cardiac myocytes.
Implications for brain stem death
As pointed out in the Introduction, a dearth of information exists on the mechanistic underpinnings of brain stem death. In a series of systematically coordinated translational research (see Chan et al. 2005b for review), our laboratory has demonstrated that the RVLM is the origin of a ‘life-and-death’ signal (Kuo et al. 1997a) that is drastically reduced or lost before patients succumb to systemic inflammatory response syndrome (Yien et al. 1997), organophosphate poisoning (Yen et al. 2000) or severe brain damage (Kuo et al. 1997b). It follows that evaluation of the biochemical changes in the RVLM during the progression towards death should shed light on the cellular and molecular mechanisms of brain stem death. Based on the same experimental endotoxaemia model used in the present study, we reported recently (Li et al. 2005) that HSP70 in this crucial neural substrate confers neuroprotection against cardiovascular fatality by enhancing the NOS I–protein kinase G signalling pathway and inhibiting the NOS II–peroxynitrite cascade. The present study provided novel findings to indicate that HSP60 in the RVLM may be another ‘pro-life’ programme in the RVLM that is activated during the progression towards brain stem death. We demonstrated that the augmented HSP60 expression confers neuroprotection against cardiovascular depression during fatal endotoxaemia via an anti-apoptotic action in the RVLM. A novel underlying mechanism which we unveiled indicated that HSP60 redistributed from mitochondrion to cytosol reduces the activation of the cytochrome c–caspase-3 cascade of apoptotic signalling by enhancing its interaction with mitochondrial or cytosolic Bax or Bcl-2. This information should provide further insights into the deterioration of central cardiovascular regulation during the progression towards brain stem death, and offer new directions for devising clinical management or therapeutic strategy against this fatal eventuality.
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