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-Dependent Oxidative Signaling Organelles in Activation-Induced T-Cell Death
ski,
Michael Kießling,
Dorothee Süss,
Peter H. Krammer, and
Karsten Gülow*
Tumor Immunology Program, German Cancer Research Center (DKFZ), Heidelberg, Germany
Received 8 December 2006/ Returned for modification 31 January 2007/ Accepted 26 February 2007
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1 (phospholipase C1), and PKC
(protein kinase C), are crucial for ROS production. PKC is translocated to the mitochondria. By using cells depleted of mitochondrial DNA, we identified the mitochondria as the source of activation-induced ROS. Inhibition of mitochondrial electron transport complex I assembly by small interfering RNA (siRNA)-mediated knockdown of the chaperone NDUFAF1 resulted in a block of ROS production. Complex I-derived ROS are converted into a hydrogen peroxide signal by the mitochondrial superoxide dismutase. This signal is essential for CD95L expression, as inhibition of complex I assembly by NDUFAF1-specific siRNA prevents AICD. Similar results were obtained when metformin, an antidiabetic drug and mild complex I inhibitor, was used. Thus, we demonstrate for the first time that PKC-dependent ROS generation by mitochondrial complex I is essential for AICD. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1 (phospholipase C1) subsequently. The activation of PLC1 results in the generation of inositol 3,4,5-triphosphate (IP3) and diacylglycerol (DAG). IP3 mediates an increase in cytosolic calcium (Ca2+), whereas DAG activates protein kinase C (PKC). The rise in cytosolic Ca2+ causes activation of the transcription factor NF-AT (nuclear factor of activated T cells) (69), one of the key regulators of CD95L expression (41). In addition, reactive oxygen species (ROS) are shown to be crucial for activation-induced CD95L expression (7, 15, 25), possibly via the ROS-inducible transcription factors NF-
B and AP-1 (17). Recently, we demonstrated that a hydrogen peroxide (H2O2)-mediated signal combined with simultaneous Ca2+ influx into the cytosol constitutes the minimal requirement for CD95L expression (25). However, the molecular source of TCR-induced ROS remains largely unclear. Aerobic organisms produce ROS by several means: in mitochondria as a by-product of respiration (63), at the endoplasmic reticulum by cytochrome P450 (50), in the cytoplasm by xanthine oxidase (20), at the plasma membrane by NADPH oxidases (35, 46) and phospholipases (54), and in peroxisomes (56). Recently, the phagocytic NADPH oxidase (NOX2) was shown to be one source for TCR-triggered ROS. However, NOX2 is not the only source for activation-induced ROS (30). Following T-cell activation, respiratory activity increases (21) and mitochondrial ROS production may be enhanced (27). In addition, there are hints supporting a possible role of the mitochondrial electron transport chain (ETC) and cytochrome P450 as origins of activation-induced ROS (7). Thus, mitochondrial involvement in activation-induced ROS generation could be addressed.
In the present study, we investigated and identified the molecular signaling pathway of TCR-induced ROS generation. Here, we demonstrate for the first time that the proximal TCR signaling machinery is essential for ROS production. Cells deficient in ZAP70, LAT, SLP76 (SH2 domain-containing leukocyte protein of 76 kDa), or PLC1 revealed no oxidative signal upon TCR stimulation. The TCR signaling machinery could be bypassed via the DAG mimetic phorbol 12-myristate 13-acetate (PMA), pointing to a role of Ca2+-independent PKCs inducing oxidative signals. Downmodulation of PKC levels by small interfering RNA (siRNA) oligonucleotides revealed that activation-induced ROS generation and CD95L expression are PKC dependent. Moreover, we demonstrate that PKC is translocated to the mitochondria and/or associated membranes upon PMA treatment. By using mitochondrial DNA (mtDNA)-depleted cells (pseudo-[rho0] cells) we show that mitochondrial function is crucial for ROS generation and subsequent AICD. Moreover, by employing specific inhibitors and siRNA-mediated knockdown of NDUFAF1, a chaperone essential for mitochondrial ETC complex I assembly (66), we demonstrate that complex I is the molecular source of activation-induced ROS in T cells. In addition, we show that the ROS produced by complex I are needed for activation of NOX2. Thus, the mitochondria are the superior source of activation-induced ROS. Finally, we prove the physiological role of complex I-induced ROS by inhibition of AICD via downmodulation of NDUFAF1 expression and application of metformin, a common antidiabetic drug and mild inhibitor of complex I (6, 18). In conclusion, the data presented here provide new insights into the molecular mechanism of AICD, suggesting a possible therapeutic role of ROS scavengers and complex I inhibitors in the treatment of CD95/CD95L-dependent disorders.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) were purchased from Calbiochem, Germany. Primary antibodies against human PKC
and PKC were supplied by BD Transduction Laboratories, Germany. Primary antibodies against human mitochondrial superoxide dismutase (MnSOD) and LAT were obtained from Upstate Biotechnology. The primary antibody against human ZnCuSOD was purchased from Santa Cruz Biotechnology, Germany. The neutralizing anti-CD95L antibody Nok1 was obtained from BD Pharmingen, Germany. All other chemicals and primary antibodies against human tubulin 
were supplied by Sigma-Aldrich, Germany. The agonistic monoclonal antibody anti-Apo-1 (mouse immunoglobulin G3), recognizing an extracellular epitope of CD95 (Apo1/Fas) (62), and the monoclonal anti-CD3 antibody OKT3 (25) were prepared as described previously.
Cell culture.
Jurkat J16-145 is a subclone of the human T-lymphoblastoid cell line Jurkat J16 (25). J.CaM2 is a LAT-negative Jurkat cell line, and J.CaM2/LAT is the control cell line retransfected with LAT (19). P116 is a ZAP70-negative Jurkat cell line (68), and P116cl.39 is the retransfected control cell line. J14 is a SLP76-deficient cell line, and J14 76-11is the retransfected control cell line (37). J.1 is a PLC1-deficient Jurkat cell line, and J.1/PLC1 is the retransfected control cell line (29). Jurkat cells were cultured in Iscove modified Dulbecco medium (IMDM) supplemented with 10% fetal calf serum (FCS).
Generation of pseudo-[rho0] cells. Cells depleted of mtDNA were generated as described previously (12, 36) with minor modifications. Briefly, Jurkat J16-145 cells were cultured in IMDM supplemented with ethidium bromide (250 ng/ml) for up to 21 days. Ethidium bromide accumulates in much higher concentrations in the mitochondrial matrix than in the nucleus. Therefore, it can be used to selectively inhibit mtDNA replication. The amount of mtDNA was examined by isolation of DNA followed by PCR specific for the mitochondrial origin of replication. The amplified product spanned the mitochondrial origin of replication of the mtDNA heavy strand between positions 15868 and 754, as follows: sense, 5'-GAAAACAAAATACTCAAATGGGCC-3'; antisense, 5'-CCTTTTGATCGTGGTGATTTAGAGGG-3'. Cells depleted of mtDNA rely energetically mainly on glycolysis and have impaired nucleotide metabolism. Therefore, pseudo-[rho0] cells were further cultured in IMDM supplemented with ethidium bromide (250 ng/ml), uridine (50 µg/ml), and sodium pyruvate (110 mg/ml). Because the cells were not completely deficient in mtDNA, they are referred to as pseudo-[rho0] cells (12). To reconstitute mtDNA content, pseudo-[rho0] cells were transferred to standard medium. Cells recovered to normal phenotype in 21 to 23 days.
Isolation of total cellular DNA. Jurkat J16-145 cells were lysed for 1 h at 55°C in 0.2 M sodium acetate-6.25% sodium dodecyl sulfate (SDS) solution containing 250 µg/ml proteinase K. Genomic DNA was isolated by phenol-chloroform extraction.
Isolation of human peripheral T cells. Human peripheral T cells were prepared by Ficoll-Paque density centrifugation, followed by rosetting with 2-amino-ethylisothyouronium-bromide-treated sheep red blood cells, as described previously (25). For activation, resting T cells were cultured at a concentration of 2 x 106 cells/ml with 1 µg/ml phytohemagglutinin for 16 h. Next, T cells were cultured in RPMI 1640 supplemented with 10% FCS and 25 U/ml interleukin 2 for 6 days (day 6 T cells), as described previously (25). All experiments were performed with T cells isolated from at least three different, healthy donors.
Isolation of human polymorphonuclear cells. Neutrophils from healthy individuals were prepared by Polymorphprep density centrifugation according to the manufacturer's instructions (Axis-Shield, Norway). All experiments were performed with neutrophils isolated from at least three different, healthy donors.
Assessment of cell death. To induce CD95L expression and/or subsequent apoptosis, cells were stimulated with anti-CD3 antibody (OKT3, 30 µg/ml) or PMA (10 ng/ml) and ionomycin (1 µM). Cell death was assessed by a drop in the forward-to-side-scatter (FSC/SSC) profile in comparison to living cells and recalculated to "specific cell death" as described previously (25).
Determination of anti-CD3- and PMA-induced ROS generation. Jurkat cells were stimulated either with plate-bound anti-CD3 (OKT3, 30 µg/ml) or with PMA (10 ng/ml) for 30 min and stained with the oxidation-sensitive dye H2DCFDA (5 µM). Since activation-induced ROS generation in human T cells was maximal at 30 min of stimulation (25), this time point was chosen for all experiments. Incubation was terminated by a wash with ice-cold phosphate-buffered saline. ROS generation was determined by fluorescence-activate cell sorting (FACS) and quantified as the increase in mean fluorescence intensity (MFI), calculated by the following formula: increase in MFI (%) = [(MFIstimulated – MFIunstimulated)/MFIunstimulated] x 100 (as described in reference 15). Cells were preincubated with inhibitors for 5 min prior to stimulation, with the exception of anti-PKC peptide inhibitors (20 min) and metformin (1 h). All experiments were performed in triplicate. The results shown are representative of at least three independent experiments.
ATP determination. Cells were lysed by freezing and thawing. Cellular ATP was measured according to the manufacturer's instructions (Molecular Probes, Germany).
Mitochondrial isolation and Western blot analysis. Crude mitochondrial fractions (containing membrane impurities) and cytoplasmic fractions were isolated with the Mitochondria Isolation Kit (Pierce), according to the manufacturer's instructions. Next, membranes were separated from mitochondria by isopycnic 0.8 to 2 M sucrose gradient centrifugation for 2 h at 80,000 x g. Figure 4A contains a schematic diagram of the purification procedure. Cells were lysed in radioimmunoprecipitation assay lysis buffer (60 mM NaCl, 25 mM Tris-HCl, 0.5% deoxycholate, 1 mM dithiothreitol, and Halt protease inhibitor cocktail [Pierce]), and the protein concentration was measured by the bicinchoninic acid assay (Pierce). SDS-polyacrylamide gel electrophoresis (PAGE) and Western blot analysis were performed as described previously (24). Band intensities were quantified by standard scanning densitometry with the NIH Image program, version 1.36b.
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Quantitative PCR. The primers and fluorescently labeled probes used for quantitative PCR were as follows: CD95L sense, 5'-AAAGTGGCCCATTTAACAGGC-3'; antisense, 5'-AAAGCAGGACAATTCCATAGGTG-3'; probe, 5'-TCCAACTCAAGGTCCATGCCTCTGG-3'; ß-actin sense, 5'-ACCCACACTGTGCCCATCTACGA-3'; antisense, 5'-CAGCGGAACCGCTCATTGCCAATGG-3'; probe, 5'-ATGCCCTCCCCCATGCCATCCTGCGT-3'. The PCR mixture (PCR kit from Eurogentech, Belgium) contained 80 µg of reverse transcribed cDNA, 1.25 ± 7.5 pM forward primers, 22.5 pM reverse primers, and 5 pM probe. For each sample, three PCRs were performed. The resulting relative increase in reporter fluorescent dye emission was monitored by the TaqMan system (GeneAmp 5700 sequence detection system and software; Perkin Elmer, Foster City, CA). The levels of the CD95L and CD95 mRNAs relative to ß-actin mRNA were calculated by the following formula: relative mRNA expression = 2–(CT of CD95L – CT of ß-actin), where CT is the threshold cycle value.
Transfection and siRNA-mediated knockdown.
Jurkat T cells and primary human T cells were transfected by lipofection (HiPerfect; QIAGEN, Germany) with negative control siRNA oligonucleotides (unlabeled or Alexa 488-labeled nonsilencing siRNA; QIAGEN, Germany) and siRNA oligonucleotides specific for human NDUFAF1 (oligo#1 antisense strand, 5'-ACUAACAUCAGGCUUCUCCdTdT-3'; oligo#2 antisense strand, 5'-UAACUAUACAUCUGAUUCGdTdT-3') or siRNA oligonucleotides specific for human PKC (Hs_PKKCD_11_HP) and PKC (Hs_PRKCQ_5_HP) (QIAGEN, Germany). Transfection was performed with 2 x 105 cells, 9 µl of transfection reagent, and different amounts of siRNA oligonucleotides ranging from 75 nM to 900 nM, according to the manufacturer's instructions. Transfected cells were rested for 48 h before being subjected to further experiments.
Measurement of MnSOD activity. MnSOD activity was determined with a commercial kit (Dojindo Molecular Technologies, Japan). Cells (1.5 x 107) were stimulated with plate-bound anti-CD3 antibody (OKT3, 30 µg/ml) or with PMA (10 ng/ml) for different time periods. Cells were harvested and lysed by freezing and thawing. The protein content was adjusted to 1 mg/ml, and SOD activity was measured with a photometer according to the manufacturer's instructions. MnSOD activity was assessed after blocking of the background activity of ZnCuSOD by the addition of 1 mM KCN to the reaction mixture.
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RESULTS |
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1 (29) were stained with DCFDA and stimulated with plate-bound anti-CD3 antibodies for 30 min. All deficient cell lines did not display any oxidative signal, whereas retransfected controls showed a clear increase of ROS upon TCR stimulation (Fig. 1A). Thus, we conclude that TCR-induced generation of ROS depends on ZAP70, LAT, SLP76, and PLC1 (Fig. 1C). As PLC1 activation results in triggering of PKCs, we investigated a possible role of PKCs in oxidative signaling by treating the deficient cell lines with PMA, a PKC activator, which bypasses ZAP70, LAT, SLP76, and PLC1. Remarkably, all deficient cell lines revealed a PMA-induced oxidative signal (Fig. 1B). PMA induces an oxidative signal without influencing the intracellular Ca2+ level (25). This implicates an involvement of Ca2+-independent PKCs in TCR-induced oxidative signaling (Fig. 1C).
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is required for activation-induced generation of ROS.
To further corroborate an involvement of PKCs in activation-induced ROS formation, cells stimulated via PMA and plate-bound anti-CD3 antibodies were pretreated with the general PKC inhibitor bisindolylmaleimide I (BIM) (Fig. 2A) or a PKC-specific peptide inhibitor (Fig. 2B). Both inhibitors blocked more than 95% of the oxidative signal. Since ROS cooperates with Ca2+ signaling for CD95L induction (25), we analyzed the impact of BIM on CD95L expression. Cells stimulated via anti-CD3 antibodies and PMA/ionomycin were pretreated with BIM. RNA was isolated, reverse transcribed, and amplified with CD95L-specific primers. In BIM-treated cells, a dose-dependent inhibition of CD95L expression was detectable (Fig. 2C). Considering that activation-induced oxidative signaling is inducible by PMA alone (Fig. 1B), we focused on Ca2+-independent novel PKC isoforms (nPKC). It has been reported that PKC, an nPKC isoform, is involved in ROS generation in keratinocytes upon overexpression (39) and in PMA/ionomycin-treated myeloid leukemia cells (43). However, despite downmodulation of PKC via siRNA, the oxidative signal was not significantly affected (<20% decrease) (Fig. 2D). This implies that PKC plays only a minor role in the generation of activation-induced ROS. PKC is unique among the nPKC isoforms because it is indispensable for T-cell development and activation (48, 59, 64). To analyze the impact of PKC on activation-induced ROS production, cells were transfected with PKC siRNA oligonucleotides (Fig. 2E). More than 80% of the PMA-induced oxidative signal was inhibited in cells transfected with PKC siRNA oligonucleotides compared to the control. These data were further confirmed by treatment of Jurkat cells with a PKC-specific peptide inhibitor, which significantly reduced the TCR- and the PMA-induced ROS levels (Fig. 2F). Moreover, siRNA-mediated downmodulation of PKC results in an inhibition of CD95L expression (Fig. 2G). Thus, we conclude that PKC is crucial for activation-induced ROS formation and CD95L expression.
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expression inhibited more than 80% of the oxidative signal (Fig. 2E), the existence of an additional PKC-dependent source of ROS in human T cells must be postulated.
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can be translocated into/to mitochondria (39, 43). To determine whether PKC is translocated to mitochondria, Jurkat cells were stimulated with PMA. PKC translocation was assessed by subjecting cytoplasmic, mitochondrial, and plasma membrane fractions to immunoblotting with anti-PKC antibodies. Surprisingly, PKC and PKC were detected in the plasma membrane fraction even in unstimulated cells. However, as expected, PKC translocates to the mitochondria after stimulation. Interestingly, the amount of PKC also increases in the mitochondrial fraction upon PMA treatment (Fig. 4A). Thus, PKC and PKC are translocated to the mitochondria and/or associated membranes in T cells after activation (Fig. 4B). In order to analyze the role of mitochondria in activation-induced ROS generation in more detail, cells transiently depleted of mtDNA, pseudo-[rho0] cells, were generated by short exposure (6 to 21 days) to small amounts of ethidium bromide (Fig. 4C) (12, 36). Upon stimulation with anti-CD3 or PMA, pseudo-[rho0] cells exhibited an up to 60% diminished oxidative signal (Fig. 4D). Since the activation-induced oxidative signal is crucial for AICD, pseudo-[rho0] cells displayed a massive reduction of AICD upon TCR stimulation and PMA/ionomycin treatment (Fig. 4E). The depletion of mtDNA was entirely reversible after removal of ethidium bromide from cell culture for 21 to 23 days (Fig. 4F). In concordance with the recovery of mitochondrial protein expression, activation-induced ROS generation (Fig. 4G) and AICD (Fig. 4H) regained normal levels. Thus, we demonstrate here that mitochondrial function is a prerequisite for induction of AICD. Complex I of the mitochondrial ETC is the source of activation-induced ROS formation. Since depletion of mtDNA leads to a decrease in activation-induced ROS generation, mtDNA-encoded proteins must be involved in oxidative signaling. Most enzymes of the ETC are oligomeric complexes consisting of both nuclear DNA- and mtDNA-encoded subunits. The primary sites for mitochondrial ROS production are complexes I and III of the ETC (45). Therefore, we aimed at analyzing the role of these complexes in activation-induced ROS production. Complex I was blocked by rotenone, a commonly used inhibitor. However, rotenone is also known to interfere with a couple of cellular pathways, including tubulin-dependent signaling events (8, 16, 32, 52). Thus, we also used a second, more specific inhibitor, piericidin A (13, 28). Complex II was inhibited by the application of 1,1,1-thenoyl trifluoroacetone (TTFA), complex III by antimycin A, and complex IV by sodium azide, and the FoF1 ATPase was blocked by oligomycin. Cells were pretreated with these inhibitors and subsequently stimulated with anti-CD3 antibodies or PMA. Thereafter, generation of ROS was determined. Only rotenone and piericidin A were able to inhibit activation-induced ROS generation, whereas TTFA, antimycin A, sodium azide, and oligomycin had no effect on or increased the oxidative signal (Fig. 5A and B). ATP levels could not account for inhibition of ROS generation, since oligomycin and antimycin A treatment resulted in a more-efficient ATP depletion than did rotenone and piericidin A (Fig. 5C). In contrast to inhibition of the NADPH oxidase (maximum 60% blockage of ROS generation [Fig. 3]), inhibition of complex I leads to a blockage of more than 95% of activation-induced ROS production (Fig. 5A and B). Therefore, complex I is not only the source of mitochondrion-derived ROS; its activity also seems to be a prerequisite for subsequent ROS production via the NADPH oxidase.
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Inhibition of complex I blocks AICD in primary human T cells. To further underline the physiological relevance of complex I-derived ROS in AICD, preactivated primary human T cells (day 6 T cells) were restimulated with anti-CD3 antibodies and pretreated with or without rotenone or antimycin A. The activation-induced oxidative signal (Fig. 9A) and CD95L expression (Fig. 9B) were inhibited exclusively by rotenone. In order to prove that complex I is the source of the oxidative signal, preactivated T cells (day 6 T cells) transfected with NDUFAF1 siRNA oligonucleotides (Fig. 9C) were used to measure ROS generation upon CD3 triggering. The NDUFAF1 siRNA oligonucleotides abolished activation-induced ROS generation by up to 70% (Fig. 9D). In addition, we analyzed the effects of metformin on primary human T cells. Metformin inhibits the anti-CD3-induced oxidative signal (Fig. 9E) and induction of CD95L expression (up to 70% inhibition) (Fig. 9F). Since AICD is mainly CD95L dependent, cell death was nearly completely inhibited by metformin (Fig. 9G). In primary T cells, apoptosis induced by direct stimulation of the CD95 receptor was not affected by metformin treatment (data not shown). Thus, the nontoxic complex I inhibitor seems to be a promising tool for treatment of diseases in which deregulation of CD95L expression plays a crucial role.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1, which generates IP3 and DAG. DAG, as well as its mimetic, PMA, activates several classes of enzymes, namely, PKCs, PKDs, DGKs, RasGRP, and chimearins (9). However, we show here an involvement of PKCs in activation-induced oxidative signaling. Application of BIM, a PKC-ATP binding blocker, and a specific pseudosubstrate peptide inhibited PMA- and TCR-induced ROS generation. PMA induces an oxidative signal without influencing the cytosolic Ca2+ level (25). Therefore, it is probable that nPKCs (calcium independent) mediate activation-induced ROS generation. Despite the fact that the nPKC isoform PKC is involved in ROS generation in keratinocytes and myeloid leukemia cells (39, 43), we show here that PKC is essential for activation-induced ROS production in T cells. Moreover, it is known that PKC is crucial for T-cell development and activation of the transcription factors AP-1 and NF-B (48, 59). These transcription factors are major regulators of CD95L expression (26). In addition, AP-1 and NF-B are ROS sensitive (17). Thus, these data are in line with the important role of PKC in oxidative signaling addressed in this study.
PKCs are known to activate NOX2. Recently, it was shown that human and murine T cells express NOX2. T cells from mice deficient in NOX2 displayed reduced ROS production upon TCR stimulation (30). Here, we demonstrate that NADPH oxidases participate in activation-induced ROS generation in human T cells. As with murine T cells, the oxidative signal is only partially NADPH oxidase dependent (Fig. 9H). Therefore, we focused on the identification of an additional source of ROS. Mitochondria are the most prominent intracellular source of ROS production (53). It has been reported that PKCs are translocated into/to mitochondria after PMA treatment (39, 43). Here, we show that upon activation PKC is translocated to the mitochondria and/or associated membrane structures. In addition, mitochondria translocate to the plasma membrane and the immunological synapse upon T-cell activation (49). To analyze the role of mitochondria in activation-induced ROS generation in more detail, we used cells transiently depleted of mtDNA, pseudo-[rho0] cells (12, 36). These cells not only reveal a diminished activation-induced oxidative signal but also a reduction in AICD. Therefore, we demonstrate for the first time that expression of mitochondrially encoded proteins is a prerequisite for induction of AICD.
The ETC components are oligomeric complexes consisting of both nuclear and mtDNA-encoded subunits. The primary sites for mitochondrial ROS production by the ETC are complexes I and III (45). It has been shown that rotenone, a commonly used inhibitor of complex I, interferes with CD8+ T-cell function (70) and activation-induced CD95L expression (7). Nevertheless, rotenone inhibits, in addition, spindle microtubule formation and tubulin assembly, leading to cell cycle arrest, disassembly of the Golgi apparatus, disturbance of the cytoskeleton, and tubulin-dependent cell-signaling events (4, 5, 8, 16, 32, 44, 52). Therefore, it is likely that rotenone interferes with formation of the immunological synapse and movement of mitochondria. However, here we prove the role of complex I in activation-induced ROS production, CD95L expression, and AICD via downmodulation of NDUFAF1 expression (Fig. 9H). Moreover, we exclude the participation of the other complexes of the ETC in activation-induced ROS production by the use of different inhibitors. Thus, we show here that it is indeed complex I that generates ROS and is therefore responsible for the induction of CD95L expression and AICD. It is discussed whether O2·– (15) or H2O2 (25) acts as a second messenger in CD95L expression and AICD. However, complex I is known to generate O2·– into the mitochondrial matrix (65). In aqueous solutions, O2·– has a half-life of less than 1 µs and is converted rapidly into H2O2 (53). MnSOD, an enzyme located the mitochondrial matrix, further facilitates the conversion of O2·– into H2O2. Here, we show that MnSOD expression and activity are enhanced upon TCR stimulation. Thus, O2·– generated by complex I is converted into H2O2 that can cross the mitochondrial membrane and act then as a second messenger in the cytosol. Blockage of complex I via inhibitors and siRNA-mediated downmodulation of NDUFAF1 expression leads to a nearly complete block of ROS generation. Therefore, complex I activity is crucial for subsequent NADPH oxidase-dependent ROS production (Fig. 9H). Recently, a similar connection between mitochondrial ROS generation and activation of NOX1 in 293T cells was described (38). Thus, we demonstrate that ROS produced by mitochondria, despite being known as damaging by-products of respiration, can also be released in a controlled process and serve as a second messenger.
Next, we searched for potential tools for manipulating the generation of ROS at complex I and verifying weather our findings have possible application in the treatment of CD95/CD95L-dependent diseases. Therefore, we analyzed the effect of metformin, an antidiabetic drug (2, 3, 58) and a mild inhibitor of complex I (6, 18), on AICD. Here, we demonstrate that metformin inhibits activation-induced ROS production and thereby CD95L expression and AICD. It has been shown in vitro that metformin inhibits reversed electron flux toward complex I (6). Therefore, we assume that activation-induced ROS production is coupled to reversed electron transport. Importantly, metformin is a nontoxic complex I inhibitor and therefore a potential tool for investigating diseases displaying defects in mitochondrial function combined with deregulation of CD95L expression.
AICD guards against the development of autoimmunity. Thus, the pathology of a recently reported case of fatal neonatal-onset mitochondrial respiratory chain disease with manifestation of T-cell immunodeficiency (51) could possibly be explained by our findings. Furthermore, multiple sclerosis (MS) is generally considered an anti-inflammatory disease with a substantial autoimmune contribution. On the one hand, it was shown in a genetic screening that about 20% of MS patients have mutations in mtDNA (34). It was also stated that mitochondrial complex I gene variants are associated with MS (67). On the other hand, many patients suffering from Leber's hereditary optic neuropathy disease, caused by mutations in the mitochondrially encoded subunits of complex I, display symptoms of MS (33). The MS pathology in patients with mutations in genes of complex I is not understood; therefore, our data warrant investigation of whether CD95L expression plays a role in its development. The same applies to the T-cell-specific immunodeficiency disorder associated with purine nucleoside phosphorylase deficiency, which is a result of the inhibition of mtDNA repair due to the accumulation of dGTP in mitochondria (1). Since CD95L plays an important role in T-cell development, mitochondrial damage may be responsible for impaired thymocyte differentiation in this disease. In addition, several T-cell-dependent diseases are associated with enhanced ROS levels, e.g., lupus erythematosus (47), rheumatoid arthritis (22), and AIDS (25), which influence T-cell activation, death, and homeostasis. Thus, the present findings may have further implications for the development of nontoxic inhibitors of complex I to treat diseases in which deregulation of CD95L expression or T-cell activation plays a vital role.
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ACKNOWLEDGMENTS |
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This project was supported by the Wilhelm Sander Stiftung, the Deutsche Forschungsgemeinschaft, and the EC.
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FOOTNOTES |
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Published ahead of print on 5 March 2007.
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0). For PCR amplification of the origin of replication of mitochondrial heavy-strand DNA (mt-ori), 100 ng of DNA template was used (upper panel). Amplification of the ß-actin gene fragment was used as a loading control (lower panel). (D) Cells depleted of mtDNA show an impaired activation-induced ROS. Parental J16-145 cells cultured in medium supplemented with uridine plus pyruvate (U+P) or cells depleted of mtDNA (ps-
) stimulated with PMA (10 ng/ml, 30 min) and cotreated with DPI (100 µM) (A) or apocynin (600 µM) (B) to inhibit the NADPH oxidase-dependent "oxidative burst." Data are presented as the FACS-measured increase in MFI of oxidized DCFDA.
