1 Alexandre Patenaude*, MRVen Murthy‡ and Marc-Edouard Mirault*§ Departments of Medicine ...

51 Pages·2004·3.09 MB·English

JBC Papers in Press. Published on April 13, 2004 as Manuscript M402496200

MITOCHONDRIAL THIOREDOXIN SYSTEM: EFFECTS OF TrxR2 OVEREXPRESSION ON
REDOX BALANCE, CELL GROWTH AND APOPTOSIS

Alexandre Patenaude*, M.R.Ven Murthy‡ and Marc-Edouard Mirault*§

Departments of Medicine* and Medical Biology‡, Faculty of Medicine, Laval University, and
CHUL/CHUQ Medical Research Center*, Québec City, Québec, G1V 4G2 Canada
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Addresses: ed
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M.-E. Mirault and A. Patenaude, Unité de santé et environnement, Centre de recherche du http
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CHUL, 2705 boulevard Laurier, Sainte-Foy, Québec, Canada G1V 4G2. w
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M.R. Ven Murthy, Dept. of Medical Biology, Faculty of Medicine, Laval University, Québec rg
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City, Québec, G1K 7P4 Canada. es
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§ Corresponding author: Marc-Edouard Mirault, Tel: [phone] ext. 47097; Fax: (418) 01
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654-2159; E-mail: [email protected]

Running title: Mitochondrial thioredoxin system and apoptosis

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Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.
The abbreviations used are: DY , mitochondrial transmembrane potential; AIF, apoptosis-
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inducing factor; ANT, adenine nucleotide translocator; BSO, buthionine sulfoximine; CCCP,
carbonylcyanide m-chlorophenylhydrazone; COX IV, cytochrome oxidase complex IV; DHR ,
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dihydrorhodamine 123; DiOC , 3’ dihexyloxacarbocyanine iodide; DTNB, 5,5’-dithiobis (2-
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nitrobenzoic) acid; EGFP, enhanced green fluorescent protein; EST, expressed sequence tag;
EtBr, ethidium bromide; etdh-1, ethidium homodimer-1; GOX, glucose oxidase; GPx,
glutathione peroxidase; Grx2, mitochondrial glutaredoxin-2; GSH, glutathione; H O , hydrogen
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peroxide; HE, dihydroethidine; MMP, mitochondrial membrane permeabilization; MnSOD,
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manganese superoxide dismutase; MTS, mitochondrial targeting sequence; MTT, 3-(4,5- o
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dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; NAD(P)H, reduced form of de
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nicotinamide adenine dinucleotide phosphate; NR, non redundant; O .-, superoxide; Prdx, h
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peroxiredoxin; ROS, reactive oxygen species; RT, room temperature; SeCys, selenocysteine; w
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STS, staurosporine; t-BHP, tert-butylhydroperoxide; TMRE, tetramethylrhodamine ethyl ester; rg
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Trx, thioredoxin; Trx2, mitochondrial thioredoxin; TrxR2, mitochondrial thioredoxin reductase- e
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2; TSA, trichostatin A; VP16, etoposide. p
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SUMMARY
Thioredoxin-2 (Trx2) is a mitochondrial protein disulfide oxidoreductase essential for control of
cell survival during mammalian embryonic development. This suggests that mitochondrial
thioredoxin reductase-2 (TrxR2), responsible for reducing oxidized Trx2, may also be a key
player in the regulation of mitochondria-dependent apoptosis. With this in mind, we investigated
the effects of overexpression of TrxR2, Trx2, or both on mammalian cell responses to various
apoptotic inducers. Stable transfectants of mouse Neuro2A cells were generated that
overexpressed TrxR2 or an EGFP-TrxR2 fusion protein. EGFP-TrxR2 was enzymatically active
and was localized in mitochondria. TrxR2 protein level and TrxR activity could be increased up D
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to 6-fold in mitochondria. TrxR2 and EGFP-TrxR2 transfectants showed reduced growth rates as de
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compared to control cells. This growth alteration was not due to cytotoxic effects nor was related h
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to changes in basal mitochondrial transmembrane potential (DY ), ROS production or to other w
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mitochondrial antioxidant components such as Trx2, peroxyredoxin-3, MnSOD, GPx-1 and rg
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glutathione whose levels were not affected by increased TrxR2 activity. In response to various e
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apoptotic inducers, the extents of DY dissipation, ROS induction, caspase activation and loss of p
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viability were remarkably similar in TrxR2 and control transfectants. Excess TrxR2 did not 0
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prevent trichostatin A-mediated neuronal differentiation of Neuro2A cells nor did it protect them
against b -amyloid neurotoxicity. Neither massive glutathione depletion nor co-transfection of
Trx2 and TrxR2 in Neuro2A (mouse), COS-7 (monkey) or HeLa (human) cells revealed any
differential cellular resistance to prooxidant or non-oxidant apoptotic stimuli. Our results suggest
that neither Trx2 nor TrxR2 gain of function modified the redox regulation of mitochondria-
dependent apoptosis in these mammalian cells.
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INTRODUCTION
A drastic alteration of the mitochondrial redox environment, which includes rapid oxidation
of NAD(P)H and glutathione is one of the earliest events of the mitochondrial pathway of
apoptosis (1-3). This phenomenon is associated with dissipation of the mitochondrial
transmembrane potential (Dy ) and subsequent induction of reactive oxygen species (ROS)
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generation (4). Onset of mitochondrial membrane permeabilization (MMP), a decisive step in the
mitochondrial pathway of apoptosis, is regulated by both pyridine nucleotide (NAD/NADH and
NADP+/NADPH) and glutathione (GSSG/GSH) redox equilibrium (5,6). Protein thiol
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(sulfhydryl) groups can be oxidized directly or indirectly by ROS, i.e. via GSH oxidation and w
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mixed disulfide formation, such as glutathiolation. For example, specific thiol groups on the d fro
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adenine nucleotide translocator (ANT), an inner membrane constituent of the mitochondrial ttp
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permeability transition pore complex, have been implicated in modulating MMP. Oxidation of w
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Cys56 and crosslinking of Cys56 to Cys159 were shown to convert the ADP/ATP translocase b/
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into an opened non-specific pore, a process causing MMP (7-9). More recently, ANT was also st o
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shown to be a critical target of apoptosis induction by nitric oxide, peroxynitrite and 4- ril 1
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hydroxynonenal (10). The cascade of events leading to MMP includes rapid NAD(P)H 1
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oxidation/depletion and tightly linked dissipation of Dy , decreased oxidative phosphorylation,
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enhanced generation of superoxide and mitochondrial osmotic swelling (4,11). Consequent
rupture of the outer mitochondrial membrane culminates with the release of soluble
intermembrane death effector proteins including the caspase-9 activator cytochrome c (12),
apoptosis-inducing factor (AIF) (13) and endonuclease G (14,15), which induce nuclear DNA
cleavage. MMP can be the rate-limiting step of the mitochondrial pathway of apoptosis,
regulated by the redox status of critical thiol groups in ANT and by interaction of the
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permeability transition pore complex with several factors including adenine nucleotides, Ca2+
and both anti-apoptotic and pro-apoptotic Bcl-2 family of proteins (reviewed in (8,9).
The mitochondrial redox environment depends on both total cellular redox environment and
compartmentalized mitochondrial reduction capacity (16). This reduction capacity depends on
the concentration of electron donor molecules such as NADPH, NADH and glutathione (GSH)
acting as antioxidant buffers, ROS detoxification enzymes and protein disulfide reductases. In
mammalian mitochondria, ROS detoxification is achieved by successive conversion of O .- into
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H O (and O ) by manganese superoxide dismutase (MnSOD) and peroxide reduction by
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glutathione peroxidases (GPx1 and GPx4) and potentially by the mitochondrial thioredoxin cycle ow
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system. Due to its elevated intracellular concentration (1-10 mM), GSH is largely responsible for d
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low redox potential and free thiol level inside cells and organelles (16,17). Experimentaly ttp
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induced glutathione deficiency in newborn rats has been shown to result in striking enlargement w
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and degeneration of mitochondria (18). A second antioxidant defense, the mitochondrial brg/
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thioredoxin system, which includes Trx2, thioredoxin reductase-2 and NADPH (see below), is a s
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potential source of disulfide reductase activity required for maintaining mitochondrial proteins in ril 1
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their reduced state; thioredoxins catalyze reduction of protein disulfides at much higher rates 01
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than GSH (19,20). An additional mitochondrial thiol/disulfide oxidoreductase, glutaredoxin-2
(Grx2), was recently discovered, which relies on GSH and not Trx as electron donor (21,22).
Little is known on the function(s) of the mitochondrial thioredoxin system and its potential
role in the regulation of cell survival. Recent studies suggest that the mitochondrial Trx system is
essential for mammalian development since disruption of Trx2 gene in the mouse confers a lethal
embryonic phenotype associated with massive apoptosis during early embryogenesis (23).
Chicken cells (DT40) conditionally deficient for Trx2 expression have been reported to undergo
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apoptosis in the absence of exogenous stress (24). In addition, glutathione depletion, serum
withdrawal and exposure to the pro-oxidant mitochondrial toxin, antimycin A, were all shown to
enhance apoptosis in Trx2 deficient DT40 cells (24,25). On the other hand, overexpression of
Trx2 was reported to enhance basal Dy and protect human HEK-293 Trx2-transfected cells
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against etoposide-mediated cytotoxicity. In contrast, these cells were not protected against
antimycin A cytotoxicity and turned out to be more sensitive than control cells to rotenone,
another pro-oxidant mitochondrial toxin (26). Another study reported that Trx2 overexpression
conferred increased oxidoresistance to human osteosarcoma cells exposed to tert-
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butylhydroperoxide (t-BHP) (26,27). Thus, the effects of Trx2 overexpression appear to be o
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complex and to depend on unidentified variables including the possibility that endogenous Trx2 de
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may not be a limiting factor in some experimental systems. h
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Peroxiredoxins (Prdx) form a new family of thiol-specific peroxidases that rely on Trx as the w
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hydrogen donor for the reduction of H2O2 and lipid hydroperoxides (28,29). Two mitochondrial b/
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Prdx isoforms have been identified so far. Prdx3, originally cloned from murine erythroleukemia st o
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cells (30), is exclusively detected in mitochondria (31). Prdx3 expression can be induced in ril 1
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response to oxidant treatments, and antisense-mediated inhibition of Prdx3 expression was 1
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shown to sensitize bovine aortic endothelial cells to oxidative challenges (32). PRDX3 was
identified as a target gene of nuclear c-Myc, and shown to be essential for maintaining
mitochondrial mass and transmembrane potential in transformed rat and human cells (33). In
addition, overexpression of Prdx3 was found to inhibit apoptosis induced by oxidative insults in
various cancer cell lines (34). Prdx5 is a second peroxiredoxin isoform that is located in the
mitochondria when expressed as a long form, while a short form was found associated with
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peroxisomes (35). Recently, Prdx5 overexpression was reported to be protective against
ibotenate-mediated excitotoxicity (36).
Mammalian TrxR isoforms are flavin homodimeric oxidoreductases with an essential redox-
active cysteine-selenocysteine (SeCys) conserved in their c-terminal part, which use NADPH to
reduce the redox-active disulfide in oxidized Trx (37,38). These enzymes can also reduce a
variety of non-disulfide substrates including selenite and peroxides. The broad substrate
specificity of TrxR appears to be restricted to the mammalian isoforms since yeast and bacterial
TrxR isoenzymes can only reduce Trx (39). A mitochondrial TrxR isoform, TrxR2, has been
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cloned from various mammals including mouse, cow, rat and human (40-45). Little is known ow
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about TrxR2 functions. The specific colocalization of TrxR2, Trx2 and Prdx3 in mitochondria, d
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presumed to function together, may suggest a defense system against H2O2 produced by the ttp
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mitochondrial respiratory chain (45). However, the relative contribution of this system to w
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peroxide detoxification, as compared to the role of the GSH system is not known. With regard to brg/
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apoptosis, it was proposed that the mitochondrial thioredoxin/thioredoxin reductase system can s
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play a role in redox regulation of the mitochondrial membrane permeability (46,47). In support ril 1
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of this suggestion, auranofin, a potent inhibitor of mitochondrial TrxR, was reported to induce 01
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MMP and loss of Dy in isolated mitochondria , two alterations that were completely inhibited by
cyclosporin A, a specific inhibitor of mitochondrial permeability transition (48). Here, we report
the effect of transfection-mediated overexpression of TrxR2 or EGFP-tagged TrxR2 on mouse
Neuro2A cell growth, mitochondrial transmembrane potential, ROS production and viability, in
response to a variety of prooxidant and non-oxidant inducers of apoptosis.
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EXPERIMENTAL PROCEDURES
Cell Culture and Reagents –Mouse Neuro2A (N2A), African green monkey COS-7 and
human HeLa cell lines were purchased from ATCC. Cells were cultured at 37(cid:176)C in 5% CO
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atmosphere in DMEM-F12 medium supplemented with 10% fetal bovine serum, 2 mM
glutamine, 100 u/ml penicillin and 100 µg/ml streptomycin (Gibco, Invitrogen), and 0.1 µM
sodium selenite. The fluorescent probes 3’ dihexyloxacarbocyanine iodide (DiOC ),
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dihydrorhodamine 123 (DHR ), dihydroethidine (HE) and tetramethylrhodamine ethyl ester
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(TMRE), were purchased from Molecular Probes. Unless specified otherwise, all reagents were
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Construction of Trx2, TrxR2, mitoEGFP and mitoEGFP-TrxR2 Expression Plasmids—Total from
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RNA was extracted from mouse liver using Trizol Reagent (Invitrogen). Full length Trx2 and ://w
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TrxR2 cDNAs were generated by RT-PCR with oligo-dT using the RETROscript kit (Ambion). c
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TrxR2 and Trx2 cDNAs were amplified by PCR with Pfu turbo polymerase using primers 83/84 g
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and 143/144, respectively (Table 1). PCR products were then isolated from agarose gels with the n A
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Qiagen gel extraction kit and subcloned in the pCR®-Blunt II-TOPO® vector (Invitrogen) before ril 11
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cloning into the EcoRI site of plasmid pCAGGS, i.e., downstream of a CMV enhancer/actin
promoter (49), to generate pCAGGS-TrxR2 or pCAGGS-Trx2. For the construction of mito-
EGFP, EGFP and the mitochondrial targeting sequence (MTS) of TrxR2 were amplified
separately, MTS from pCAGGS-TrxR2 with primers 83/216, and EGFP from pEGFP-N1
(Clontech) with primers 217/219 including a stop codon. The amplified MTS and EGFP DNAs
were digested with Nhe1, ligated together and digested by EcoRI. Following separation of
ligation products in agarose gels, DNA was isolated from the band of correct size (0.87 kb) and
inserted in the EcoRI site of pCAGGS. For the construction of mitoEGFP-TrxR2, MTS was
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amplified from pCAGGS-TrxR2 with primers 83/216, EGFP from pEGFP-N1 with primers
217/218, and TrxR2 excluding MTS from pCAGGS-TrxR2 with primers 215/84. Amplified
MTS and EGFP were digested by NheI, EGFP and TrxR2 DNA products were digested by SacI,
and the three DNA preparations mixed together for ligation. Following separation of ligation
products in agarose gels, DNA was isolated from the band of correct size (2.6 kb) and inserted
in the EcoRI site of pCAGGS. Recombinant plasmids with the required correct insert orientation
were identified by restriction enzyme digestion. This was confirmed by DNA sequencing of
whole recombinant DNAs inserted in pCAGGS.
Transfections—Neuro2A transfectant cells expressing exogenous TrxR2, Trx2 or EGFP D
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fusion proteins to various extents were obtained by calcium phosphate transfections (50) using de
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increasing ratios of pCAGGS constructs to pINDsp1-Hygro plasmid (for selection with h
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hygromycin-B, Invitrogen). The basal amount of pINDsp1-Hygro was always 1 µg per 100 mm w
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culture dish (Sarstedt). Stable transfectant populations were generated with the following .org
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pCAGGS constructs using pCAGGS/pINDsp1-Hygro µg DNA ratios as indicated in brackets: ue
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CtrlH63, pCAGGS (30/1); TrxR2H56, pCAGGS-TrxR2 (10/1); TrxR2H64, pCAGGS-TrxR2 (30/1); pril 1
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Trx2 , pCAGGS-Trx2 (30/1); Trx2/TrxR2 , pCAGGS-Trx2 (30/1) and pCAGGS-TrxR2 , 2
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(30/1); TrxR2 , pCAGGS-TrxR2 (60/1); mito-EGFP , pCAGGS-mito-EGFP (30/1),
H163 H143
EGFP-TrxR2 , pCAGGS-EGFP-TrxR2 (30/1). Selection for hygromycin-B (500µg/ml)
H144
resistant cells started 48 h post-transfection for at least two weeks. Resistant colonies from each
transfection were pooled to produce a heterogeneous population of transfected cells. The clone
TrxR2 derives from one colony isolated from a pCAGGS-TrxR2 transfectant population.
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Fugene 6 reagent (Roche) was used for transient transfections according to procedure
recommended by the manufacturer. In brief, a ratio of 3/1 for Fugene reagent (µl) / plasmid (µg)
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was incubated for 30 min at RT in incomplete medium before addition to 50% sub-confluent
cells in complete medium for a period of 24 h.
Cell Fractionation—Cell fractionation was carried out by differential centrifugation mainly as
described previously (51). In brief, cells (2-4 x 107) were dounced with 60 strokes with a tight
fitting pestle in buffered sucrose (BS): 20 mM Hepes pH 7.5, 250 mM sucrose, 1 mM EDTA, 1
mM EGTA, 10 mM KCl and a tablet/10 ml of complete mix protease inhibitors (Roche). After
two centrifugations at 1,000 g to discard nuclei, mitochondria were pelleted at 10,000 g, washed
once and resuspended in BS before storage at -80(cid:176)C. The cytosolic fraction was obtained by
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centrifugation of the post-mitochondrial supernatant at 100,000 g for 1 h. Protein contents were o
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determined by Bradford assay (Biorad). de
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Antibodies and Western Blot Analysis—Polyclonal antibodies against mouse Trx2, Prdx3 and h
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TrxR2 were produced by injecting rabbits with hemocyanin-conjugated peptides, w
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CLEAFLKKLIG for Trx2, CSPTASKEYFEKVHQ for Prdx3 and KRSGLEPTVTGCCG for rg
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TrxR2 respectively. The peptides were injected in complete Freund’s adjuvant for the first e
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immunization and with incomplete adjuvant six weeks later for three immunization boosts p
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administered every three weeks. Affinity purified GPx-1 antibody was prepared from a rabbit , 20
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antiserum produced by repeated immunization with recombinant human GPx1 expressed in
bacteria (52), with three boosts at two-week intervals followed by three boosts at one month
intervals using bovine GPx protein (Sigma G-6137). The antibody was purified by affinity for
bovine GPx isolated by SDS-PAGE gel and electrotransfer on PVDF membrane. All antibodies
were stored at -20(cid:176)C after addition of 50 % glycerol. SDS -PAGE (10%) was performed with
equal amounts of protein (10 µg per track) separated in mini gels (Mini-Protean II, Biorad) and
electrotransferred on to PVDF membranes. The membranes were saturated with TBS-0.1%
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