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. 2007 May;27(10):3625-39.
doi: 10.1128/MCB.02295-06. Epub 2007 Mar 5.

Novel role for mitochondria: protein kinase Ctheta-dependent oxidative signaling organelles in activation-induced T-cell death

Marcin Kaminski  1 , Michael Kiessling, Dorothee Süss, Peter H Krammer, Karsten Gülow
Affiliations

Novel role for mitochondria: protein kinase Ctheta-dependent oxidative signaling organelles in activation-induced T-cell death

Marcin Kaminski et al. Mol Cell Biol. 2007 May.

Abstract

Reactive oxygen species (ROS) play a key role in regulation of activation-induced T-cell death (AICD) by induction of CD95L expression. However, the molecular source and the signaling steps necessary for ROS production are largely unknown. Here, we show that the proximal T-cell receptor-signaling machinery, including ZAP70 (zeta chain-associated protein kinase 70), LAT (linker of activated T cells), SLP76 (SH2 domain-containing leukocyte protein of 76 kDa), PLCgamma1 (phospholipase Cgamma1), and PKCtheta (protein kinase Ctheta), are crucial for ROS production. PKCtheta 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 PKCtheta-dependent ROS generation by mitochondrial complex I is essential for AICD.

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Figures

FIG. 1.
FIG. 1.
Activation-induced ROS generation depends on the proximal TCR signaling machinery. (A and B) Jurkat J16-145, P116 (ZAP70-negative Jurkat), P116cl.39 (ZAP70-retransfected control), J.CaM2 (LAT-negative Jurkat), J.CaM2/LAT (LAT-retransfected control), J14 (SLP76-deficient Jurkat), J14 76-11 (SLP76-retransfected control), J.γ1 (PLCγ1-deficient Jurkat), and J.γ1/PLCγ1 (PLCγ1-retransfected control) cells were stimulated via plate-bound anti-CD3 antibodies (A) or with PMA (B) for 30 min. Thereafter, cells were stained with DCFDA. Representative FACS profiles for activation-induced DCFDA oxidation are shown. ctr, retransfected control, def. cell lines, lines deficient in signaling molecules. (C) Schematic diagram of TCR signaling.
FIG. 2.
FIG. 2.
PKCθ is required for activation-induced generation of ROS. (A) J16-145 cells were pretreated with the indicated amounts of the PKC inhibitor BIM and stimulated with PMA (left) or plate-bound anti-CD3 antibodies (right) for 30 min. Cells were stained with DCFDA and analyzed by FACS. Data shown as the percent increase in MFI. (B) J16-145 cells were pretreated with the indicated amounts of a general PKC pseudosubstrate peptide inhibitor, stained with DCFDA, and stimulated with PMA (left) or plate-bound anti-CD3 antibodies (right) for 30 min. ROS levels were measured as for panel A. (C) J16-145 Jurkat cells were pretreated with the indicated amounts of the PKC inhibitor BIM and stimulated with PMA/ionomycin (left) or plate-bound anti-CD3 antibodies (right). After 1 h, RNA was isolated, reverse transcribed, and amplified with CD95L- and actin-specific primers. (D) Jurkat J16-145 cells were transfected with 900 nM concentrations of scrambled (Ctr) or PKCδ siRNA (PKCδ) oligonucleotides. After 96 h, transfected cells were lysed and analyzed by Western blotting for content of PKCδ (right) or stained with DCFDA, stimulated via PMA for 30 min, and subjected to FACS analysis (left); results are shown as the percent increase in MFI. (E) Jurkat J16-145 cells were transfected with 900 nM concentrations of scrambled (Ctr) or PKCθ siRNA (PKCθ) oligonucleotides. At 96 h after transfection, cells were analyzed as described for panel D. Shown are Western blots for PKCθ content (left) and PMA-induced DCFDA oxidation (right). (F) Jurkat J16-145 cells were pretreated with PKCθ pseudosubstrate peptide inhibitor and stimulated with PMA (left) or by plate-bound anti-CD3 antibodies (right) for 30 min. DCFDA oxidation was measured by FACS and presented as the increase in MFI. (G) Jurkat cells were transfected with scrambled (ctr) or PKCθ siRNA (PKCθ) oligonucleotides (as described for panel E). Cells were stimulated with PMA/ionomycin. After 1 h, RNA was isolated, reverse transcribed, and amplified with CD95L- and actin-specific primers.
FIG. 3.
FIG. 3.
Activation-induced ROS generation is partially NADPH oxidase dependent. Jurkat J16-145 cells were pretreated with the NADPH oxidase inhibitor DPI (A) or apocynin (B), stained with DCFDA, and stimulated with PMA for 30 min. Inserts show human neutrophils (NΦ) 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.
FIG. 4.
FIG. 4.
PKCθ is translocated toward mitochondria upon PMA treatment. (A) J16-145 cells were stimulated with PMA for 10 min. Cells were lysed and cellular fractions were separated as depicted in the diagram (S1, S2, respective supernatants; P1, P2, respective pellets). Highlighted fractions were separated by SDS-PAGE and analyzed by Western blotting for content of PKCθ, PKCδ, ZnCuSOD (cytoplasmic marker), MnSOD (mitochondrial marker), and LAT (plasma membrane marker). (B) Schematic diagram of PKCθ translocation and ROS induction. (C to H) Involvement of mtDNA-encoded proteins in activation-induced ROS generation and AICD. (C) Total cellular DNA was isolated from parental J16-145 cells, J16-145 cells cultured in the presence of uridine (50 μg/ml) and pyruvate (110 mg/ml) (U+P), and J16-145 cells cultured in the presence of uridine plus pyruvate and ethidium bromide (250 ng/ml) (ps-ρ0). 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 β-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-ρ0) were stimulated via plate-bound anti-CD3 antibodies (left) or with PMA (right) for 30 min, stained with DCFDA, and analyzed by FACS. The percent increase in MFI is shown. (E) Cells depleted of mtDNA show lowered AICD. Cells (U+P or ps-ρ0) as described for panel C were stimulated via plate-bound anti-CD3 antibodies or with PMA/ionomycin. After 24 h, cell death was measured by a drop in the FSC/SSC index and results were recalculated to specific cell death. (F) The content of mtDNA was tested for parental J16-145 cells or pseudo-[rho0] cells, which regained mtDNA after long-term culture due to withdrawal of ethidium bromide from the culture medium (recov). Total cellular DNA (100 ng) was used and amplified as described for panel C. (G) Parental Jurkat J16-145 cells cultured in standard medium or pseudo-[rho0] cells after recovery of mtDNA content (recov) were stimulated by plate-bound anti-CD3 antibodies (left) or with PMA (right) for 30 min, and activation-induced ROS production was measured as described for panel D. (H) Parental J16-145 or recovered (recov) cells were stimulated by plate-bound anti-CD3 antibodies (left panel) or with PMA/ionomycin (right panel). After 24 h, cell death was determined as described for panel E.
FIG. 5.
FIG. 5.
Complex I of the mitochondrial ETC is the source of activation-induced ROS formation. (A and B) Jurkat J16-145 cells were pretreated with the indicated amounts of ETC inhibitors (ROT, rotenone; Pier, piericidin A; AA, antimycin A; TTFA, 1,1,1-thenoyl trifluoroacetone; Az, sodium azide) or an inhibitor of the FoF1 ATPase (OLI, oligomycin), stained with DCFDA, stimulated by PMA (A) or plate-bound anti-CD3 antibody (B) for 30 min, and analyzed by FACS. The data are presented as the percent increase in MFI. (C) Jurkat J16-145 cells were treated with high concentrations of inhibitors of the ETC or the FoF1 ATPase for 2 h. Thereafter, cells were lysed and ATP content was determined. (D) Mitochondrion-derived ROS induce changes in expression and activity of MnSOD. Jurkat J16-145 cells were stimulated with plate-bound anti-CD3 antibodies or PMA/ionomycin (Iono) for the indicated time periods. Isolated RNA was reverse transcribed and amplified with MnSOD-specific primers. (E) Jurkat cells were stimulated via plate-bound anti-CD3 antibodies or PMA/ionomycin (Iono) for the indicated time points. Cells were lysed and MnSOD protein levels were determined by Western blot analysis. MnSOD expression was normalized to tubulin and quantified with NIH Image (lower panel). (F) MnSOD activity in mitochondria of J16-145 cells stimulated by plate-bound anti-CD3 antibody or PMA.
FIG. 6.
FIG. 6.
ROS produced by complex I drive activation-induced CD95L expression. (A and B) J16-145 cells were pretreated with the indicated amounts of inhibitors of the ETC (ROT, rotenone; Pier, piericidin A; AA, antimycin A; TTFA, 1,1,1-thenoyl trifluoroacetone; Az, sodium azide) or the FoF1 ATPase (OLI, oligomycin) and stimulated with PMA/ionomycin (Iono) (A) or plate-bound anti-CD3 antibodies (B) for 1 h. RNA was isolated, reverse transcribed, and amplified with CD95L- and actin-specific primers. (C) J16-145 cells were pretreated with the indicated inhibitors and stimulated with (left) or without (right) plate-bound anti-CD3 antibodies for 1 h. RNA was isolated and reverse transcribed, and a quantitative PCR was performed. CD3-induced CD95L expression was set to 100%. All other values were calculated according to the CD3-induced CD95L expression. (D) Schematic diagram of mitochondrial ROS production. CI, complex I.
FIG. 7.
FIG. 7.
Downregulation of NDUFAF1 inhibits ROS generation, CD95L expression, and AICD. (A) J16-145 cells were transfected with 75 nM concentrations of scrambled (ctr) or two different NDUFAF1-siRNA (#1 and #2) oligonucleotides. After 48 h, RNA was isolated, reverse transcribed, and amplified with NDUFAF1- and actin-specific primers. (B) At 48 h after transfection with scrambled- (ctr) or NDUFAF1-siRNA (#1 and #2) oligonucleotides, the oxidative signal upon 30 min of PMA treatment was determined by DCFDA staining (filled profile, stained cells/untreated; open profile, cells stained and stimulated with PMA). (C) Quantification of PMA-induced oxidative signals in Jurkat cells at 72 h after transfection with 75 nM concentrations of scrambled (ctr) or NDUFAF1-siRNA (#1 and #2) oligonucleotides. Cells were stained with DCFDA, treated with PMA for 30 min, and subjected to FACS analysis. Results are shown as the percent increase in MFI. (D) J16-145 cells were transfected with 75 nM concentrations of scrambled (ctr) or NDUFAF1-siRNA (#1 and #2) oligonucleotides. After 72 h of resting, cells were treated with PMA/ionomycin for 1 h. PMA/ionomycin RNA was isolated, reverse transcribed, and amplified with CD95L- and actin-specific primers. (E) J16-145 cells were transfected with 75 nM (left) or 900 nM (right) concentrations of scrambled (ctr) or NDUFAF1-siRNA (#2) oligonucleotides. After 72 h of resting, AICD was induced by 24 h of PMA/ionomycin treatment. Cell death was assessed by a drop in the FSC/SSC index. Results were recalculated to specific cell death.
FIG. 8.
FIG. 8.
Metformin, a nontoxic complex I inhibitor, blocks activation-induced oxidative signal, CD95L expression, and AICD. (A) Metformin induces no toxicity. J16-145 cells were treated for 2 h (white bars) and 24 h (gray bars) with the indicated inhibitors (ROT, rotenone [10 μg/ml]; Pier, piericidin A [7.5 μM]; Metf, metformin [100 μM]; TTFA, 1,1,1-thenoyl trifluoroacetone [25 μM]; AA, antimycin A [4 μg/ml]; Az, sodium azide [100 μg/ml]; OLI, oligomycin [10 μg/ml]). Cell death was determined by a drop in the FSC/SSC profile in comparison to living cells and recalculated to specific cell death. (B) J16-145 cells were pretreated with the indicated amounts of metformin, stained with DCFDA, and stimulated by PMA for 30 min. Oxidative signal was quantified as the increase in MFI. (C and D) J16-145 cells were pretreated with the indicated amounts of metformin and stimulated with PMA/ionomycin (Iono) (C) or plate-bound anti-CD3 antibodies (D) for 1 h. Next, RNA was isolated, reverse transcribed, and amplified with CD95L- and actin-specific primers (left). In addition, a quantitative PCR was performed (right). CD3-induced CD95L expression was set to 100%. All other values were calculated according to the CD3-induced CD95L expression. (E and F) J16-145 cells pretreated with the indicated amounts of metformin and AICD were induced by PMA/ionomycin (Iono) treatment (E) or stimulation with plate-bound anti-CD3 antibodies (F). After 24 h, cell death was assessed by the drop in the FSC/SSC index. Inserts show J16-145 cells cotreated with or without CD95L neutralizing antibody (NOK1) and stimulated by plate-bound anti-CD3 antibodies. Results were recalculated to specific cell death.
FIG. 9.
FIG. 9.
Primary human T cells depend on complex I-originated activation-induced ROS for CD95L expression and AICD. (A) T cells were pretreated with the indicated amounts of inhibitors of the ETC (ROT, rotenone; AA, antimycin A) and stimulated with anti-CD3 antibodies for 30 min. Cells were stained with DCFDA, and the increase in MFI was measured by FACS. (B) T cells were pretreated with the indicated amounts of rotenone (upper panel) or antimycin A (lower panel) and stimulated with anti-CD3 antibodies for 1 h. Next, RNA was isolated, reverse transcribed, and amplified with CD95L- and actin-specific primers. (C) T cells were transfected with 900 nM scrambled (ctr) or two different NDUFAF1-siRNA (#1 and #2) oligonucleotides. After 48 h, RNA was isolated, reverse transcribed, and amplified with NDUFAF1- and actin-specific primers. (D) At 72 h after transfection with 900 nM scrambled (ctr) or NDUFAF1-siRNA (#1 and #2) oligonucleotides, primary human T cells were stimulated by plate-bound anti-CD3 antibodies for 30 min and the oxidative signal was determined as for panel A. (E to G) T cells were pretreated with the indicated amounts the nontoxic complex I inhibitor metformin and stimulated with plate-bound anti-CD3 antibodies (i) for 30 min (E), to measure the oxidative signal (quantified as in panel A); (ii) for 1 h (F), to detect changes in CD95L expression (left, semiquantitative PCR; right, quantitative PCR); or (iii) for 24 h (G), to asses AICD by a drop in the FCS/SSC index. The insert shows T cells cotreated with or without CD95L neutralizing antibody (NOK1); results were recalculated to specific cell death. (H) Schematic diagram of TCR-induced oxidative signaling.
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