Modulation of Mitochondrial Transition Pore Components by Thyroid Hormone
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内分泌学杂志 2005年第5期
Department of Human Nutrition and Metabolism, Hebrew University Medical School, Jerusalem 91120, Israel
Address all correspondence and requests for reprints to: Dr. Jacob Bar-Tana, Department of Human Nutrition and Metabolism, Hebrew University Medical School, Jerusalem 91120, Israel. E-mail: bartanaj@cc.huji.ac.il.
Abstract
Thyroid hormone (TH) modulates metabolic efficiency by controlling the coupling of mitochondrial oxidative phosphorylation. However, its uncoupling mode of action is still enigmatic. Treatment of Jurkat or GH3 cells with T3 is reported here to result in limited, Cyclosporin A-sensitive mitochondrial depolarization, conforming to low conductance gating of the mitochondrial transition pore (MTP). MTP protein components induced by T3 treatment were verified in T3-treated and hypothyroid rat liver as well as in Jurkat cells. T3 treatment resulted in increase in mitochondrial Bax and Bak together with decreased mitochondrial Bcl2. T3-induced mitochondrial depolarization was aborted by overexpression of Bcl2. In contrast to Bax-Bcl2 family proteins, some other MTP components were either not induced by T3 (e.g. voltage-dependent anion channel) or were induced, but were not involved in Cyclosporin A-sensitive MTP gating (e.g. Cyclophilin D and adenine nucleotide translocase-2) Hence, TH-induced mitochondrial uncoupling may be ascribed to low conductance MTP gating mediated by TH-induced increase in mitochondrial proapoptotic combined with a decrease in mitochondrial antiapoptotic proteins of the Bax-Bcl2 family.
Introduction
THYROID HORMONE (TH) has long been recognized as a major modulator of mitochondrial metabolic efficiency. However, its mitochondrial mode of action is still enigmatic (reviewed in Refs. 1 and 2). Short-term mitochondrial effects of TH are refractory to inhibitors of protein synthesis, whereas the long-term effect is dependent on gene expression. Short-term direct uncoupling of mitochondrial oxidative phosphorylation due to the protonophoric capacity of TH is questionable in light of the nonphysiological T3 levels required (3, 4). Short-term uncoupling by binding of T3 or diiodothyroinine to specific mitochondrial components [e.g. adenine nucleotide translocase (ANT) and Cytochrome c oxidase] is still controversial (5). The long-term effect induced by TH is mediated by T3 binding to T3 receptors of the superfamily of nuclear receptors, resulting in direct transcriptional activation of the expression of genes coding for mitochondrial oxidative phosphorylation components (i.e. ?F1-adenosine triphosphatase, ANT, and Cytochrome c1) (1, 6) or genes coding for intermediate factors (i.e. nuclear respiratory factors 1 and -2, and peroxisome proliferator-activated receptor- coactivator-1) indirectly involved in the nuclear expression of mitochondrial components (reviewed in Ref. 7). The long-term effects of TH in the mitochondrial context are also complemented by stimulating mitochondrial DNA replication and expression by T3 receptor-induced mitochondrial transcription factor or by activating mitochondrial truncated forms of c-erbA (reviewed in Ref. 8). However, the respective genes and proteins directly involved in TH-induced mitochondrial uncoupling remain to be identified. The proposed candidates include protein components of mitochondrial channels [e.g. uncoupling protein-1 (UCP1) to UCP-3, with UCP3 being preferentially favored due to its expression in skeletal muscle) (reviewed in Ref. 9). However, reports concerned with correlations among the response to TH, the extent of UCP3 expression, and the extent of mitochondrial uncoupling are conflicting (reviewed in Ref. 9), thus calling for additional putative targets for TH-induced mitochondrial uncoupling.
The mitochondrial transition pore (MTP) spans the contact sites of the inner and outer mitochondrial membranes (reviewed in Ref. 10). The MTP composition is still questionable. It is reported to consist of ANT in the inner mitochondrial membrane in juxtaposition with the voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane, complemented by Cyclophilin D and members of the Bax-Bcl2 family of proteins. MTP gating is initiated by Ca2+-triggered conformational changes in MTP components (11), facilitated by binding of mitochondrial matrix Cyclophilin D to MTP-ANT (12, 13). Liver mitochondria lacking ANT may still be induced to undergo permeability transition if exposed to high Ca2+ (14), thus indicating that ANT is not an essential structural component of liver mitochondrial MTP, but does contribute to its gating regulation. Ca2+ triggering of MTP gating may be enhanced by oxidative stress, depletion of adenine nucleotides, increased inorganic phosphate, increased matrix pH, depolarization of the inner mitochondrial membrane, or increased fatty acids (reviewed in Refs. 15, 16, 17). Under energized conditions, the pore is also sensitive to ANT ligands, being activated by Atractylate and inhibited by Bongrekic acid (reviewed in Ref. 18). Cyclosporin A acts as a potent inhibitor of MTP gating due to its binding to Cyclophilin D and interferes with its interaction with MTP-ANT (19 ; but see Refs. 20 and 21).
MTP gating may present itself in variable modes, differing in reversibility and synchronization. Definitive synchronized MTP gating, induced by intramitochondrial Ca2+ load under conditions that facilitate Ca2+ binding to MTP-ANT, results in extensive depolarization of the inner mitochondrial membrane, rapid passage of ions and solutes of less than 1500 Da across the inner mitochondrial membrane, and mitochondrial swelling, which may result in rupture of the outer mitochondrial membrane (reviewed in Ref. 22). The release of matrix proapoptotic proteins, such as Cytochrome c, apoptotic intrinsic factor and others, may initiate programmed cell death/apoptosis. Alternatively, spontaneous, nonsynchronized, transient/flickering MTP gating due to cyclic opening and closure of individual MTP channels may result in reversible and limited depolarization of the inner mitochondrial membrane, moderate decrease in proton motive force (μH), and passage of solutes of less than 300 Da accompanied by mitochondrial contraction rather than swelling (23, 24, 25). Transient/low conductance MTP (LC-MTP) gating is innocuous, in contrast to the irreversible proapoptotic depolarization inflicted by definitive MTP gating.
MTP gating has been previously reported by us to be modulated by TH treatment. Thus, in vivo T3 treatment resulted in a 70% decrease in mitochondrial membrane potential (), proton gradient (pH), and μH of Ca2+-loaded rat liver mitochondria accompanied by their extensive swelling. The effect of T3 under conditions of mitochondrial Ca2+ loading was essentially eliminated by added Cyclosporin A, thus pointing to its MTP context (26, 27). These studies have been confirmed in rat liver mitochondria of hypothyroid rats, where Cyclosporin A-sensitive mitochondrial Ca2+ efflux, swelling, and release of matrix proteins were all impaired even under conditions of Ca2+ loading (28, 29), but were restored by T3 treatment (28, 30). These findings have prompted us to verify the TH effect in T3-responsive cells and to identify mitochondrial MTP components, induced or suppressed by in vivo T3 treatment, that may be functionally involved in MTP gating by TH.
Materials and Methods
Animals
Male albino rats, weighing 150–180 g, were fed a standard laboratory chow diet (diet 19520, Koffolk, Tel Aviv, Israel). Hyperthyroidism was induced by daily injections of L-T3 as indicated, in 0.05 N NaOH in saline for 5 d. Control animals were injected with the vehicle only. Hypothyroidism was induced by 0.025% (wt/vol) methimazole in the drinking water for 4 wk. Serum T3 levels were measured when the animals were killed, 20 h after the last T3 injection, by RIA (kit from Diagnostic Products Corp., Los Angeles, CA) and were 0.6 ± 0.2, 3.9 ± 0.5, 3.5 ± 0.4 and 15 ± 3 nM in hypothyroid rats, euthyroid rats, and rats treated with 2 and with 50 μg T3/100 g body weight (BW), respectively. Fed animals from all groups were killed between 0800–0900 h by injection of 0.1 ml/100 g BW of a mixture containing 85 mg/ml ketamine and 3 mg/ml xylazine. The experimental protocol was approved by the ethical committee for animal and human experimentation of Hebrew University.
Mitochondria isolation
Rat liver mitochondria were isolated as previously described with slight modifications (31). Livers were homogenized in 300 mM sucrose, 20 mM Tris-HCl (pH 7.4), 2 mM EGTA, 0.1 μg/ml phenylmethylsulfonylfluoride, 1 μg/ml leupeptin, and 0.7 μg/ml pepstatin, followed by centrifugation at 1,100 x g for 10 min at 4 C. The supernatants were filtered through gauze and additionally centrifuged at 1,100 x g for 10 min at 4 C, followed by centrifugation at 10,800 x g for 10 min at 4 C for precipitating the crude mitochondrial fraction. Mitochondrial matrix was prepared as described previously (32).
Cells
Jurkat cells stably transfected with pEF-puro-Bcl2 expression plasmid or with the respective empty vector, as described by Huang et al. (33), were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 11 mM HEPES, 22 mM glutamine, 100 U penicillin/ml, and 0.1 mg streptomycin/ml. For T3 treatment, grown Jurkat cells were cultured in RPMI 1640 medium containing 10% charcoal-stripped FCS (Biological Industries, Beit Hemeek, Israel), followed by culture in RPMI 1640 medium containing 10–1000 nM T3 for the indicated time periods. One half volume of the culture medium was exchanged every other day. Where indicated, Cyclosporin A (4 μg/ml) was added to the culture 48 h before analysis.
Rat pituitary GH3 cells were grown in 30-mm culture dishes in MEM (Biological Industries) supplemented with 10% FCS, 0.1% glucose, and 20 mM glutamine. Cells were depleted of T3 using charcoal-stripped FCS. For TH treatment, 100–500 nM T3 and/or 7.5 μg/ml Cyclosporin A were added as indicated to the culture medium for 48 h before analysis.
HeLa and COS-7 cells were cultured in DMEM (Biological Industries) supplemented with 10% FCS, 50 U penicillin/ml, and 50 μg streptomycin/ml. Cells grown to 50% confluence on round glass coverslips, 12 mm in diameter, were transiently transfected using the calcium-phosphate precipitation with pcDNA3-green fluorescence protein (pcDNA3-GFP; 0.5 μg), pcDNA3-ANT2 (0.5–2.0 μg), or pcDNA3-Cyclophilin D (0.2–3.0 μg DNA) as indicated. After 6 h of incubation with the DNA precipitate, the cells were washed twice with PBS and cultured in fresh medium.
Mitochondrial membrane potential ()
Twenty-four-hour-transfected HeLa cells were incubated for 30 min in DMEM containing 150 nM chloromethyl-X-rosamine (MitoTracker red, Molecular Probes, Eugene, OR) at 37 C in the dark. Cells were washed with Hanks’ balanced salt solution and fixed in 3% paraformaldehyde for 10 min. The was determined by confocal microscopy (LSM 410, Carl Zeiss, Inc., New York, NY) by measuring the MitoTracker red intensity of GFP-transfected cells compared with neighboring, nontransfected cells, using ImagerPro Plus software (Media Cybernetics, Silver Spring, MD).
GH3 cells were incubated for 30 min in MEM containing 150 nM MitoTracker red at 37 C in the dark, followed by replacing the medium with Hanks’ balanced salt solution. The of living, nonfixed cells was immediately determined by confocal microscopy by measuring the MitoTracker red intensity of images of T3-treated cells compared with untreated cells.
Jurkat cells (1 x 106 cells) were incubated at 37 C in the dark for 30 min in RPMI 1640 medium containing 40 nM 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3); Molecular Probes] (34). Cells were centrifuged at 1000 x g for 2 min, and the pellet was washed once by resuspension in 400 μl PBS. The of washed cells was immediately analyzed by flow cytometric scanning (FACS; FL-2 setting; 20,000 events/analysis) using CellQuest software (BD Biosciences, Mountain View, CA). Uncoupling by carbonyl cyanide m-chlorophenylhydrazone (CCCP) was used as a positive control.
Cyclophilin D immunostaining
HeLa cells transfected with Cyclophilin D were fixed in 3% paraformaldehyde for 10 min and permeabilized by 0.1% Triton X-100 in a buffer containing 50 mM Tris-HCl (pH 7.6), 0.15 M NaCl, and 3% (wt/vol) BSA for 25 min. Permeabilized cells were washed and incubated with the same buffer for an additional 30 min for blocking nonspecific binding, followed by reacting the cells with rabbit antiCyclophilin D for 3 h and with secondary antirabbit Cy5 antibody for 1 h at room temperature.
Western blot analysis
Respective cells (1 x 107 to 3 x 107/sample) or liver tissue were homogenized by a hand-held glass homogenizer or by Polytron (Brinkmann Instruments, Westbury, NY), respectively, in 3 vol lysis buffer containing PBS, 1% Triton, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1 mM phenylmethylsulfonylfluoride, 10 μg leupeptin/ml, and 1 μg aprotinin/ml. Isolated mitochondria were suspended in lysis buffer as described above. After incubation in lysis buffer for 30 min at 4 C, respective lysates were cleared by centrifugation at 15,000 x g for 30 min, and supernatants were kept at –70 C. Equivalent amounts of proteins (80 μg) were resolved by 12.5% SDS-PAGE under reducing conditions and were transferred onto polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA) or cellulose nitrate membranes (Schleicher & Schuell, Dassel, Germany). Blots were probed with the indicated first antibody, followed by horseradish peroxidase-labeled second antibody. Bands were analyzed by ImageQuant software (Molecular Devices, Sunnyvale, CA).
Northern blot analysis
Total RNA was prepared from rat livers using the Ez-RNA extraction kit (Biological Industries). RNA (25 μg) was electrophoresed in a 1% formaldehyde agarose gel, blotted onto nylon membrane (GeneScreen Plus, NEN Life Science Products, Boston, MA), and cross-linked at 80 C for 2 h. Cyclophilin D mRNA was probed by Northern blot hybridization with 620 bp Cyclophilin D cDNA prepared by RT-PCR from rat total RNA using the sense 5'-CTAGGACAGCAGCAGGCAGC-3' and antisense 5'-TTGAGCAGACAGGCCTGGCT-3' primers (U68544 in GenEMBL cDNA bank). Blots were visualized and quantitated by phosphorimager densitometry (BAS 1000, FUJIX, Tokyo, Japan).
Materials
Jurkat cells (stably transfected with Bcl2 or with the empty vector) were obtained from G. Hacker (Technische Universitat, Munich, Germany). pcDNA3-Cyclophilin D and pcDNA3-ANT2 expression vectors were obtained from S. Grimm (Max Planck Institute for Biochemistry, Martinsried, Germany). Cyclosporin A was from Novartis Pharmaceuticals (East Hanover, NJ). Rabbit polyclonal antirat and antihuman Bcl2 antibody (SC-492, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), rabbit antirat and antihuman Cyclophilin D antibody (PA1–028, Affinity BioReagents, Golden, CO), rabbit antirat VDAC antibody (PA1–954, Affinity BioReagents), mouse monoclonal antirat and antihuman Bak antibodies (AM04, Oncogene Research Products, San Diego, CA) and rabbit antihuman Bax antibody (SC-493, Santa Cruz Biotechnology, Inc.) were commercially available. Rabbit antirat Bax antibody was obtained from Atan Gross (Weizmann Institute of Science, Rehovot, Israel). Horseradish peroxidase-labeled antirabbit and antimouse secondary antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA).
Data analysis
Statistical analysis was performed by one-way repeated measure ANOVA with Student-Newman-Keuls test. When only two groups were compared, significance was analyzed by paired t test.
Results
MTP gating by T3
MTP gating by T3 in Jurkat cells has been evaluated by measuring mitochondrial of cells cultured in the presence or absence of added T3 for up to 22 d. The was measured by flow cytometry, using the -sensitive dye DiOC6(3), in the absence and presence of added Cyclosporin A. As shown in Fig. 1A and in line with findings previously reported by Mihara et al. (34), most Jurkat cells (90%) were characterized by high fluorescence, reflecting their polarized inner . A minor population (10%) consisted of low fluorescent cells, reflecting their collapsed . Treatment with variable T3 concentrations (10–1000 nM) for 14–22 d resulted in a limited (13–17%), but significant, Cyclosporin A-sensitive mitochondrial depolarization of the major cell population (Fig. 1). Depolarization was already saturated by 10 nM T3, and an effect of lower T3 concentrations cannot be ruled out. Culturing of Jurkat cells with T3 for 7 d did not result in mitochondrial depolarization. Cyclosporin A-sensitive mitochondrial depolarization of Jurkat cells by T3 treatment was accompanied by a 33% increase in the minor low cell population (Figs. 1A and 9D; see below).
FIG. 1. LC-MTP gating induced by T3 in Jurkat cells. Jurkat cells were cultured for 14–22 d, as described in Materials and Methods, in the absence or presence of T3 or Cyclosporin A as indicated. Cultured cells were stained with DiOC6(3) and analyzed by FACS as described in Materials and Methods. A, Representative flow cytometric analysis of in untreated, T3-treated (500 nM), or T3 (500 nM) and Cyclosporin A (4.0 μg/ml)-treated cells. B, Histogram summarizing the effect of increasing concentrations of T3 (0–1000 nM) in the absence or presence of Cyclosporin A on the mean fluorescence intensity of the highly stained (channels, 2 x 102 to 3 x 103; A) cell population. The mean fluorescence of untreated cells is defined as 1.0. Values shown are the mean ± SE of three independent experiments performed in triplicate. *, Significant compared with untreated cells (P < 0.05).
FIG. 9. Bcl2 protection of T3-induced mitochondrial depolarization. Jurkat cells transfected with Bcl2 or with vector alone (WT) were grown for 14–22 d in the absence or presence of T3 as indicated. Cultured cells were stained with DiOC6(3) and analyzed by FACS as described in Materials and Methods. A, The Bcl2 protein content of nontransfected and Bcl2-transfected Jurkat cells determined by Western blot analysis as described in Materials and Methods. B, Representative flow cytometric analysis of of untreated and T3 (500 nM)-treated Jurkat cells, transfected with Bcl2 (right panel) or with vector alone (left panel). C, Histogram summarizing the mean fluorescence intensity of untreated and T3-treated Jurkat cells transfected with Bcl2 () or with vector alone () and sampled during d 14–22. Values shown are the mean ± SE of three independent experiments. *, Significant compared with the respective untreated cells (P < 0.05). D, Histogram summarizing the percentage of low cells of untreated and T3-treated (500–1000 nM; see Fig. 7) Jurkat cells transfected with Bcl2 () or with vector alone () and sampled during d 14–22. Values shown are the mean ± SE of three independent experiments. *, Significant compared with non-T3-treated, non-Bcl2-transfected cells (P < 0.05). #, Significant, by paired t test, compared with non-T3-treated, non-Bcl2-transfected cells (P < 0.05).
MTP gating by T3 has been verified as well in GH3 cells by confocal microscopy using MitoTracker red. As shown in Fig. 2, culturing GH3 cells with T3 for 48 h resulted in Cyclosporin A-sensitive, T3-dependent mitochondrial depolarization, amounting to a 28% decrease in . These results in Jurkat and GH3 cells complement our previous studies using Ca2+-loaded liver mitochondria derived from T3-treated rats, where T3 treatment induced a Cyclosporin A-sensitive, 70% decrease in mitochondrial pH and (26).
FIG. 2. LC-MTP gating induced by T3 in GH3 cells. GH3 cells were cultured for 48 h in the absence or presence of T3 and Cyclosporin A as indicated, followed by staining with MitoTracker red as described in Materials and Methods. A, Representative confocal microscopic images of untreated and T3-treated cells. B, Histogram summarizing the fluorescence intensity of MitoTracker red of untreated and T3-treated (500 nM) cells in the absence or presence of Cyclosporin A. Each column represents the mean fluorescence ± SE of 50–70 images collected from three independent experiments. *, Significant compared with untreated cells (P < 0.05). #, Significant compared with T3-treated cells (P < 0.05).
T3-induced MTP components
Cyclosporin A-sensitive MTP gating induced by T3 treatment in rat liver mitochondria (26, 28) or in cells (Figs. 1 and 2) was investigated in terms of the effects of T3 on the expression and function of MTP protein components.
ANT2.
ANT2 is essentially the only ANT isoform of rat liver mitochondria and the only isoform induced by T3 treatment (35). The role of ANT2 in MTP gating was evaluated by measuring Cyclosporin A-sensitive depolarization of the of HeLa cells overexpressing ANT2. Cells expressing ANT2 were identified by their cotransfected GFP. As shown in Fig. 3A, overexpression of ANT2 resulted in decreased , as verified by MitoTracker red images of transfected compared with neighboring nontransfected cells. The remained unaffected in GFP-transfected cells in the absence of transfected ANT2. The extent of depolarization induced by overexpressed ANT2 was dependent on the ANT2 transfection load. Thus, transfection of cells with low ANT2 (0.5 μg) did not affect , whereas higher ANT2 (>2.0 μg) induced an average 40% decrease in (Fig. 3B), indicating that T3-induced ANT2 could putatively account for T3-induced uncoupling. However, mitochondrial depolarization induced by overexpressed ANT2 was not affected by Cyclosporin A (Fig. 3B), indicating that Cyclosporin A-sensitive MTP gating induced by T3 is not mediated by T3-induced ANT2. Results similar to those derived by MitoTracker red imaging were verified by measuring mitochondrial depolarization by tetramethylrhodamine (TMRM) (not shown).
FIG. 3. T3-induced ANT2 in MTP gating. A, Representative confocal microscopic images of HeLa cells transfected with expression plasmids for GFP (0.5 μg; blue image) together with pcDNA3 (3.0 μg; upper panel) or pcDNA3-ANT2 (3.0 μg; lower panel) and stained with MitoTracker red (green image). Note the depolarization specifically induced by ANT2 in cells cotransfected with GFP and ANT2. B, Histogram summarizing the ratio between the fluorescence intensity of MitoTracker red of cells transfected with variable concentrations of pcDNA3-ANT2 and neighboring nontransfected cells. Where indicated, Cyclosporin A (CSA; 7.5 μg/ml) was added to the culture 24 h before MitoTracker red staining. The fluorescence intensity ratio of cells transfected with pcDNA3 is defined as 1.0. Values shown are the mean ± SE of 150 images collected from eight independent experiments. *, Significant compared with pcDNA3-transfected cells (P < 0.05).
VDAC.
The effect of T3 on liver mitochondrial VDAC levels was analyzed by measuring VDAC protein content in liver mitochondria isolated from untreated or T3-treated euthyroid or hypothyroid rats (Fig. 4). Mitochondrial VDAC levels remained essentially unaffected by T3, indicating that MTP gating induced by T3 treatment is not mediated by T3-induced VDAC.
FIG. 4. Effect of T3 on mitochondrial VDAC protein level. The mitochondrial VDAC protein contents of euthyroid (E), hyperthyroid (T), hypothyroid (H), and T3-treated hypothyroid (HT) rats were determined by Western blot analysis as described in Materials and Methods. The top panel illustrates a representative gel. Densitometric intensity of euthyroid VDAC is defined as 1.0. Values shown are the mean ± SE of four rats.
Cyclophilin D.
Cyclophilin D transcript and protein levels were assayed in livers of euthyroid, T3-treated, hypothyroid and T3-treated, hypothyroid rats. T3 treatment resulted in 2-fold increase in liver Cyclophilin D transcript (Fig. 5A) and protein (Fig. 5B) levels, whereas hypothyroidism resulted in a significant 2-fold decrease in liver Cyclophilin D protein, which was fully restored by T3 treatment (Fig. 5B). T3-induced Cyclophilin D was enzymatically functional, as verified by the increased peptidyl-prolyl cis-trans isomerase activity in T3-treated rat liver mitochondria (Fig. 5C).
FIG. 5. T3-induced Cyclophilin D. A, Cyclophilin D transcript relative to 28S rRNA of liver extracts from euthyroid (E) and T3-treated (T) rats determined by Northern blot analysis. The densitometric intensity of euthyroid Cyclophilin D mRNA is defined as 1.0. Values shown are the mean ± SE of four rats. *, Significant compared with euthyroid rats (P < 0.05). B, Cyclophilin D protein content of liver extracts derived from euthyroid (E), T3-treated (T), hypothyroid (H), and T3-treated hypothyroid (HT) rats determined by Western blot analysis. The densitometric intensity of euthyroid cells is defined as 1.0. The top panel illustrates a representative gel. Values shown are the mean ± SE of four rats. *, Significant compared with euthyroid rats (P < 0.05). #, Significant compared with hypothyroid rats (P < 0.05). C, The peptidyl-prolyl cis-trans isomerase (PPIase) activity of liver mitochondrial matrix prepared from euthyroid (E) and T3-treated (T) rats was assayed as described previously (37 ) in the absence or presence of added Cyclosporin A (CSA; 7.5 μg/ml). The initial rate of PPIase activity of euthyroid mitochondrial matrix is defined as 1.0. Values shown are the mean ± SE of seven rats. +, Average of two experiments differing by less than 10%. *, Significant compared with euthyroid rats (P < 0.05).
The effect of increased Cyclophilin D levels on MTP gating was evaluated by analyzing the of HeLa cells overexpressing Cyclophilin D. HeLa cells overexpressing Cyclophilin D were identified by cotransfection with GFP or by Cy5-labeled antiCyclophilin D antibody. The of cells transfected or not transfected with Cyclophilin D was verified by MitoTracker red intensity compared with neighboring nontransfected cells. Cyclophilin D overexpression could be expected to result in decreased and increased MTP sensitivity to oxidative stress (10). Surprisingly, however, MitoTracker red intensity was maintained or even increased in cells overexpressing Cyclophilin D (Fig. 6, A and B), indicating that the remained essentially unaffected or was even hyperpolarized by overexpressed Cyclophilin D. Mitochondrial colocalization of MitoTracker red and Cyclophilin D was verified by the yellow overlay image of mitochondrial MitoTracker red (Fig. 6A, green image) and transfected Cyclophilin D (Fig. 6A, red image). The intramitochondrial localization of transfected Cyclophilin D was verified by analyzing the processed/intramitochondrial (18-kDa) and unprocessed/extramitochondrial (21-kDa) Cyclophilin D proteins (36) in Cyclophilin D-transfected COS-7 cells. As shown in Fig. 6C, most of the transfected Cyclophilin D was in its processed form (18 kDa), indicating its intramitochondrial localization. Results similar to those derived by MitoTracker red imaging were verified by measuring mitochondrial depolarization with TMRM (not shown).
FIG. 6. T3-induced Cyclophilin D in MTP gating. A, Representative confocal microscopic images of HeLa cells transfected with expression plasmids for GFP (0.5 μg; blue image) together with pcDNA3 (3.0 μg; upper panel) or pcDNA3-Cyclophilin D (3.0 μg; lower panel). Transfected cells were stained with MitoTracker red (green image) and immunostained with antiCyclophilin D antibody, followed by Cy5-labeled secondary antibody (red image). Colocalization of mitochondrial MitoTracker red and Cy5-labeled Cyclophilin D results in a yellow image, indicating Cyclophilin D targeting to mitochondria. B, Histogram summarizing the fluorescence intensity of MitoTracker red of HeLa cells transfected with increasing pcDNA3-Cyclophilin D. The fluorescence intensity of cells transfected with pcDNA3 is defined as 1.0. Values shown are the mean ± SE of 150 images collected from eight independent experiments. *, Significant as compared with pcDNA3-transfected cells (P < 0.05). C, Western blots of pcDNA3-transfected (left lane) and pcDNA3-Cyclophilin D (6 μg)-transfected (right lane) COS-7 cells stained for Cyclophilin D.
The lack of effect of overexpressed Cyclophilin D on mitochondrial MTP gating was verified under conditions of oxidative stress induced by tert-butyl hydroperoxide. Tert-butyl hydroperoxide-induced mitochondrial depolarization in cells overexpressing Cyclophilin D was similar to that in control cells (not shown). These results may indicate that LC-MTP gating by T3 is not accounted for by T3-induced Cyclophilin D.
Bcl2 family components.
The family of Bax-Bcl2 proteins is grouped into two main subfamilies: proapoptotic proteins (e.g. Bax, Bak, and others) and antiapoptotic proteins (e.g. Bcl2), which promote or inhibit MTP gating, respectively (reviewed in Refs. 37 and 38). The effect of T3 on the expression of representative proteins of the two subfamilies was investigated in livers of euthyroid; T3-treated, hypothyroid; and T3-treated, hypothyroid rats as well as in Jurkat cells. Treatment of euthyroid or hypothyroid rats with T3 induced a dose-dependent increase in liver mitochondrial Bax (Fig. 7A) and resulted in 3-fold increase in liver mitochondrial Bak (Fig. 7B). In contrast to Bax and Bak, liver mitochondrial Bcl2 was increased by 20-fold in hypothyroid rats and was decreased by 35-fold in T3-treated, hypothyroid rats (Fig. 8A). The reciprocal changes in Bax and Bak contrasted with those in Bcl2 resulted in 5- and 7.5-fold respective increases in liver mitochondrial Bax/Bcl2 and Bak/Bcl2 ratios in T3-treated animals and 20- and 25-fold respective decreases in liver mitochondrial Bax/Bcl2 and Bak/Bcl2 ratios in hypothyroid rats. These results were corroborated in Jurkat cells, where T3 treatment for 14–22 d resulted in a dose-dependent 50% increase in cellular Bax (Fig. 7C) together with a 30% decrease in Bcl2 levels (Fig. 8B) (34) concomitant with their T3-induced, Cyclosporin A-sensitive mitochondrial depolarization (Fig. 1). No significant effect of T3 on Bax or Bcl2 content was observed in Jurkat cells incubated in the presence of T3 for 7 d, in line with the 14- to 22-d period required for their T3-induced, Cyclosporin A-sensitive mitochondrial depolarization (Fig. 1).
FIG. 7. T3-induced Bax and Bak. A, Mitochondrial Bax protein content of euthyroid (E), hyperthyroid (50 μg T3/l00 g BW; T), hypothyroid (H), and T3-treated hypothyroid (HT) rats. The densitometric intensity of euthyroid cells is defined as 1.0. Values shown are the mean ± SE of four rats. Inset, Histogram of liver mitochondrial Bax content of hypothyroid rats treated with increasing T3 doses as indicated. The densitometric intensity of hypothyroid cells is defined as 1.0. Values shown are the mean ± SE of four rats. The top panels illustrate representative gels. *, Significant compared with euthyroid rats (P < 0.05). #, Significant compared with hypothyroid rats (P < 0.05). B, Mitochondrial Bak protein content of euthyroid (E), hyperthyroid (50 μg T3/l00 g BW; T), hypothyroid (H), and T3-treated (50 μg T3/l00 g BW) hypothyroid (HT) rats. The top panel illustrates a representative gel. Densitometric intensity of euthyroid cells is defined as 1.0. Values shown are the mean ± SE of four rats in each treatment group. *, Significant compared with euthyroid rats (P < 0.05). #, Significant compared with hypothyroid rats (P < 0.05). C, The Bax protein content of untreated Jurkat cells and cells treated with T3 for 22 d as indicated. The top panel illustrates a representative gel of cells treated with increasing T3 concentrations as indicated. The densitometric intensity of untreated Jurkat cells is defined as 1.0. Values are the mean ± SE of three independent experiments performed in triplicate. Because the T3-induced decrease in is saturated by T3 concentrations higher than 10 nM (Fig. 1B), the results of two experiments using 500 nM T3 and one experiment using 1000 nM T3 were grouped for statistical analysis. *, Significant compared with untreated cells (P < 0.05).
FIG. 8. Suppression of Bcl2 by T3. A, Mitochondrial Bcl2 protein contents of euthyroid (E), hyperthyroid (50 μg T3/l00 g BW; T), hypothyroid (H), and T3-treated (50 μg T3/l00 g BW) hypothyroid (HT) rats were determined by Western blot analysis as described in Materials and Methods. The top panel illustrates a representative gel. The densitometric intensity of euthyroid Bcl2 is defined as 1.0. Values shown are the mean ± SE of four rats. *, Significant compared with euthyroid rats (P < 0.05). #, Significant compared with hypothyroid rats (P < 0.05). B, The Bcl2 protein content of untreated and 22-d T3-treated Jurkat cells determined by Western blot analysis as described in Materials and Methods. The top panel illustrates a representative gel of cells treated with increasing T3 concentrations as indicated. The densitometric intensity of untreated Jurkat Bcl2 is defined as 1.0. Values shown are the mean ± SE of three independent experiments performed in triplicate using 500-1000 nM T3 (see Fig. 7). *, Significant compared with untreated cells (P < 0.05).
The changes observed in liver mitochondrial content of Bax-Bcl2 proteins were only partly accounted for by respective changes in the total cellular content of the proteins concerned. Thus, the 2.9-fold increase in liver mitochondrial Bak (Fig. 7B) was correlated with a similar 3.0 ± 0.4-fold (P < 0.05) increase in total cellular Bak content, indicating that the T3-induced increase in mitochondrial Bak could be accounted for by its increased expression level. However, the 2.6-fold decrease in liver mitochondrial Bcl2 induced by T3 treatment (Fig. 8A) was in contrasted to the T3-induced 3.4 ± 0.7-fold (P < 0.05) increase in total cellular Bcl2 (not shown), indicating that suppression of liver mitochondrial Bcl2 by T3 treatment resulted from T3-induced factors other than its mere expression level.
The induction/suppression of Bax-Bcl2 family proteins by T3 in the context of MTP gating was evaluated in Jurkat cells overexpressing Bcl2 (Fig 9A). T3 treatment resulted in a Cyclosporin A-sensitive, 13–17% decrease in the of the major cell population (Fig. 1, A and B, and Fig. 9, B and C) accompanied by a 33% increase in the minor population of low cells (Fig. 9, B and D). However, overexpression of Bcl2 resulted in essentially eliminating T3-induced mitochondrial depolarization of the high cell population (Fig. 9, B and C) as well as eliminating the T3-induced increase in the low cell population (Fig. 9, B and D). Hence, suppression of mitochondrial Bcl2 combined with induction of mitochondrial Bax and Bak by T3 treatment may account for MTP gating by TH.
Discussion
TH-induced MTP components
Mitochondrial depolarization induced by T3 in Jurkat and GH3 cells, as reported here, conforms to LC-MTP gating. MTP involvement is indicated by its sensitivity to Cyclosporin A, whereas the limited extent of mitochondrial depolarization induced by T3 conforms to low conductance permeability transition. These findings complement those of our previous study, where T3 treatment in vivo combined with mitochondrial Ca2+ loading in vitro resulted in definitive Cyclosporin A-sensitive MTP gating, leading to extensive mitochondrial depolarization and mitochondrial swelling (26).
MTP gating induced by T3 was pursued in this study in terms of MTP components induced by T3 treatment, which could account for sensitization to MTP gating. T3 treatment is reported to result in pronounced increases in liver mitochondrial Bax and Bak together with a pronounced decrease in Bcl2, whereas hypothyroidism resulted in opposite effects that were reversed by T3. Also, the kinetics of T3-induced MTP gating in Jurkat cells was correlated with those of changes in their contents of respective Bax-Bcl2 proteins. Because the resultant effect of Bax-Bcl2 proteins on MTP gating is determined by the ratio between pro- and antiapoptotic proteins (39, 40), the effect of T3 is amplified by oppositely modulating pro- and antiapoptotic proteins of the Bax-Bcl2 family. Moreover, protection of T3-induced MTP gating by overexpressed Bcl2 may indicate that respective Bax-Bcl2 proteins induced/suppressed by T3 treatment transduce MTP gating induced by T3. MTP gating has indeed been repeatedly reported to be positively or negatively affected by proapoptotic (e.g. Bax and Bak) and antiapoptotic (e.g. Bcl2) proteins of the Bcl2 family, respectively (reviewed in Refs. 37 and 38). Bax-Bcl2 family proteins may directly interact with other MTP components such as ANT (41, 42) or VDAC (43), and when overexpressed or added to isolated mitochondria, they may specifically induce (e.g. Bax and Bak) (44, 45, 46) or antagonize (e.g. Bcl2) (47) MTP gating. Similarly, depletion of proapoptotic Bax or Bak results in failure of MTP gating (41, 47, 48), whereas Bcl2 inactivation results in definitive MTP gating triggered by oxidative stress (49).
The mode of action of T3 in enriching/depleting mitochondrial members of the Bax-Bcl2 family remains to be investigated in terms of the expression of nuclear and mitochondrial genes putatively involved in translocating and anchoring Bax-Bcl2 proteins to the MTP complex of specific cell types. The T3-induced increase in total liver Bcl2, in contrast with its mitochondrial depletion (see Results), may indicate that T3-induced factors other than expression levels of Bax-Bcl2 proteins (e.g. their phosphorylation and mitochondrial translocation) are involved in modulating the liver mitochondrial content of respective MTP components.
In contrast to proteins of the Bax-Bcl2 family, TH-induced MTP gating does not seem to be transduced by TH-induced ANT2 or Cyclophilin D. In light of its structural (10, 13) and/or regulatory (14) functions in the MTP complex, ANT2 expression induced by T3 treatment (35) could, in principle, account for liver MTP gating induced by T3. Overexpression of mitochondrial ANT2 was indeed found here to result in extensive mitochondrial depolarization, similar to that previously reported for overexpressed ANT1 (50, 51, 52). The discrepancy between our findings and the previously reported lack of effect of overexpressed ANT2 on mitochondrial depolarization and apoptosis (50, 52) could result from differences in ANT2 transfection yields or its mitochondrial targeting. However, mitochondrial depolarization induced by overexpressed ANT2 was insensitive to Cyclosporin A (Fig. 3B), reflecting the formation of an MTP-nonrelated ANT channel (53) or of Cyclosporin A-insensitive MTP (21) by overexpressed ANT2. The lack of an obligatory linkage between MTP and ANT conforms to recent findings pointing to MTP gating by proapoptotic ligands in liver cells and isolated mitochondria lacking ANT altogether (14). These findings taken together with the lack of effect of T3 treatment on VDAC levels (Fig. 4) imply that T3-induced, Cyclosporin A-sensitive MTP gating is not accounted for by modulating the mitochondrial content of ANT or VDAC.
Induction of Cyclophilin D by T3 treatment could similarly be expected to result in Cyclosporin A-sensitive MTP gating in light of the putative role of Cyclophilin D in modulating ANT affinity for Ca2+, which is aborted by Cyclosporin A (10, 12, 13). In vivo T3 treatment was indeed found here to induce Cyclophilin D transcript, protein, and peptidyl-prolyl cis-trans isomerase activities. Surprisingly, however, overexpression of Cyclophilin D and its mitochondrial targeting resulted in mitochondrial hyperpolarization, rather than MTP gating. Our results do not agree with those previously reported by De Giorgy et al. (54), but corroborate those presented by Lin et al. (55), who reported that was hyperpolarized in cells overexpressing Cyclophilin D. Furthermore, our results are in line with previous reports that overexpressed Cyclophilin D desensitized cells to apoptotic stimuli or to protect cells from mitochondrial depolarization and apoptosis induced by overexpression of ANT1 (50, 51, 55). Hence, induction of Cyclophilin D by T3 may not account for T3-induced MTP gating.
MTP gating by TH
Our previously (26, 27) and presently reported results may indicate that in vivo TH treatment results in low conductance MTP or definitive MTP gating, depending on a variety of physiological or pathological signals. The two MTP modes have distinct characteristics and may serve distinct functions. Thus, TH-induced LC-MTP gating represents an innocuous limited mitochondrial depolarization (23, 24, 25) and is proposed to control metabolic efficiency by modulating the extent of coupling of mitochondrial oxidative phosphorylation. TH has indeed long been realized to increase mitochondrial mass and oxygen consumption together with decreasing the mitochondrial phosphate potential and mitochondrial redox state (56, 57). Mitochondrial uncoupling by TH is exerted at the level of the mitochondria, as verified by the stimulation of state 4 respiration, increase in proton leak, and decrease in membrane potential of mitochondria isolated from TH-treated rats (58, 59). Because mitochondrial uncoupling accounts for 25% and 50% of state 4 respiration in liver and muscle, respectively, and the phosphorylating machinery competes with mitochondrial proton leaks for the same driving force, the overall enthalpy required for carrying out a given task may be dominated by TH-induced LC-MTP gating under both basal (57) as well as working (59) conditions.
In contrast to LC-MTP gating, TH-induced definitive MTP gating results in extensive mitochondrial depolarization and swelling mediated by synchronized, irreversible MTP opening, leading to apoptosis (37, 38). TH-induced apoptosis of peripheral blood T lymphocytes has indeed been exemplified in vivo in Graves’ disease patients (34). The definitive MTP gating mode may serve the role of organ development, exemplified by TH-induced tadpole tail apoptosis in the course of amphibian metamorphosis (60, 61), or, alternatively, may pose a risk under conditions of cardiac ischemia-reperfusion damage (62, 63).
TH treatment is proposed to promote the LC and definitive gating modes of MTP by enriching/depleting mitochondrial Bax-Bcl2 proteins. Changes in mitochondrial Bax-Bcl2 proteins may affect spontaneous MTP gating frequency, gating synchronization of individual pores, or sensitivity to Ca2+ or oxidative stress. The increase in the number of low Jurkat cells induced by T3 concomitantly with LC-MTP gating of the major cell population (Fig. 1) and the abrogation of both by overexpressed Bcl2 (Fig. 9) indicate that LC-MTP gating induced by T3 may promote drift into definitive MTP gating (Fig 10). Hence, depending on the prevailing triggering signals, TH-sensitized MTP may either be operating in its restrained physiological constraints in the context of LC-MTP gating or drift nonreversibly into definitive MTP gating.
FIG. 10. MTP gating by T3. The T3-induced increase in the Bax/Bcl2 ratio is proposed to drive MTP gating from its closed hypothyroid configuration to its low conductance hyperthyroid configuration, resulting in limited depolarization and progressive decoupling of mitochondrial oxidative phosphorylation with concomitant increase in calorigenesis. Exposure of T3-sensitized MTP to oxidative stress or increased mitochondrial Ca2+ may result in definitive MTP gating, leading to collapsed and apoptosis.
Acknowledgments
The kind generosity of G. Hacker (Jurkat cells), S. Grimm (pcDNA-ANT2 and Cyclophilin D expression vectors), A. Gross (rabbit antirat Bax antibody), and Novartis Pharmaceuticals (Cyclosporin A) is deeply acknowledged.
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Address all correspondence and requests for reprints to: Dr. Jacob Bar-Tana, Department of Human Nutrition and Metabolism, Hebrew University Medical School, Jerusalem 91120, Israel. E-mail: bartanaj@cc.huji.ac.il.
Abstract
Thyroid hormone (TH) modulates metabolic efficiency by controlling the coupling of mitochondrial oxidative phosphorylation. However, its uncoupling mode of action is still enigmatic. Treatment of Jurkat or GH3 cells with T3 is reported here to result in limited, Cyclosporin A-sensitive mitochondrial depolarization, conforming to low conductance gating of the mitochondrial transition pore (MTP). MTP protein components induced by T3 treatment were verified in T3-treated and hypothyroid rat liver as well as in Jurkat cells. T3 treatment resulted in increase in mitochondrial Bax and Bak together with decreased mitochondrial Bcl2. T3-induced mitochondrial depolarization was aborted by overexpression of Bcl2. In contrast to Bax-Bcl2 family proteins, some other MTP components were either not induced by T3 (e.g. voltage-dependent anion channel) or were induced, but were not involved in Cyclosporin A-sensitive MTP gating (e.g. Cyclophilin D and adenine nucleotide translocase-2) Hence, TH-induced mitochondrial uncoupling may be ascribed to low conductance MTP gating mediated by TH-induced increase in mitochondrial proapoptotic combined with a decrease in mitochondrial antiapoptotic proteins of the Bax-Bcl2 family.
Introduction
THYROID HORMONE (TH) has long been recognized as a major modulator of mitochondrial metabolic efficiency. However, its mitochondrial mode of action is still enigmatic (reviewed in Refs. 1 and 2). Short-term mitochondrial effects of TH are refractory to inhibitors of protein synthesis, whereas the long-term effect is dependent on gene expression. Short-term direct uncoupling of mitochondrial oxidative phosphorylation due to the protonophoric capacity of TH is questionable in light of the nonphysiological T3 levels required (3, 4). Short-term uncoupling by binding of T3 or diiodothyroinine to specific mitochondrial components [e.g. adenine nucleotide translocase (ANT) and Cytochrome c oxidase] is still controversial (5). The long-term effect induced by TH is mediated by T3 binding to T3 receptors of the superfamily of nuclear receptors, resulting in direct transcriptional activation of the expression of genes coding for mitochondrial oxidative phosphorylation components (i.e. ?F1-adenosine triphosphatase, ANT, and Cytochrome c1) (1, 6) or genes coding for intermediate factors (i.e. nuclear respiratory factors 1 and -2, and peroxisome proliferator-activated receptor- coactivator-1) indirectly involved in the nuclear expression of mitochondrial components (reviewed in Ref. 7). The long-term effects of TH in the mitochondrial context are also complemented by stimulating mitochondrial DNA replication and expression by T3 receptor-induced mitochondrial transcription factor or by activating mitochondrial truncated forms of c-erbA (reviewed in Ref. 8). However, the respective genes and proteins directly involved in TH-induced mitochondrial uncoupling remain to be identified. The proposed candidates include protein components of mitochondrial channels [e.g. uncoupling protein-1 (UCP1) to UCP-3, with UCP3 being preferentially favored due to its expression in skeletal muscle) (reviewed in Ref. 9). However, reports concerned with correlations among the response to TH, the extent of UCP3 expression, and the extent of mitochondrial uncoupling are conflicting (reviewed in Ref. 9), thus calling for additional putative targets for TH-induced mitochondrial uncoupling.
The mitochondrial transition pore (MTP) spans the contact sites of the inner and outer mitochondrial membranes (reviewed in Ref. 10). The MTP composition is still questionable. It is reported to consist of ANT in the inner mitochondrial membrane in juxtaposition with the voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane, complemented by Cyclophilin D and members of the Bax-Bcl2 family of proteins. MTP gating is initiated by Ca2+-triggered conformational changes in MTP components (11), facilitated by binding of mitochondrial matrix Cyclophilin D to MTP-ANT (12, 13). Liver mitochondria lacking ANT may still be induced to undergo permeability transition if exposed to high Ca2+ (14), thus indicating that ANT is not an essential structural component of liver mitochondrial MTP, but does contribute to its gating regulation. Ca2+ triggering of MTP gating may be enhanced by oxidative stress, depletion of adenine nucleotides, increased inorganic phosphate, increased matrix pH, depolarization of the inner mitochondrial membrane, or increased fatty acids (reviewed in Refs. 15, 16, 17). Under energized conditions, the pore is also sensitive to ANT ligands, being activated by Atractylate and inhibited by Bongrekic acid (reviewed in Ref. 18). Cyclosporin A acts as a potent inhibitor of MTP gating due to its binding to Cyclophilin D and interferes with its interaction with MTP-ANT (19 ; but see Refs. 20 and 21).
MTP gating may present itself in variable modes, differing in reversibility and synchronization. Definitive synchronized MTP gating, induced by intramitochondrial Ca2+ load under conditions that facilitate Ca2+ binding to MTP-ANT, results in extensive depolarization of the inner mitochondrial membrane, rapid passage of ions and solutes of less than 1500 Da across the inner mitochondrial membrane, and mitochondrial swelling, which may result in rupture of the outer mitochondrial membrane (reviewed in Ref. 22). The release of matrix proapoptotic proteins, such as Cytochrome c, apoptotic intrinsic factor and others, may initiate programmed cell death/apoptosis. Alternatively, spontaneous, nonsynchronized, transient/flickering MTP gating due to cyclic opening and closure of individual MTP channels may result in reversible and limited depolarization of the inner mitochondrial membrane, moderate decrease in proton motive force (μH), and passage of solutes of less than 300 Da accompanied by mitochondrial contraction rather than swelling (23, 24, 25). Transient/low conductance MTP (LC-MTP) gating is innocuous, in contrast to the irreversible proapoptotic depolarization inflicted by definitive MTP gating.
MTP gating has been previously reported by us to be modulated by TH treatment. Thus, in vivo T3 treatment resulted in a 70% decrease in mitochondrial membrane potential (), proton gradient (pH), and μH of Ca2+-loaded rat liver mitochondria accompanied by their extensive swelling. The effect of T3 under conditions of mitochondrial Ca2+ loading was essentially eliminated by added Cyclosporin A, thus pointing to its MTP context (26, 27). These studies have been confirmed in rat liver mitochondria of hypothyroid rats, where Cyclosporin A-sensitive mitochondrial Ca2+ efflux, swelling, and release of matrix proteins were all impaired even under conditions of Ca2+ loading (28, 29), but were restored by T3 treatment (28, 30). These findings have prompted us to verify the TH effect in T3-responsive cells and to identify mitochondrial MTP components, induced or suppressed by in vivo T3 treatment, that may be functionally involved in MTP gating by TH.
Materials and Methods
Animals
Male albino rats, weighing 150–180 g, were fed a standard laboratory chow diet (diet 19520, Koffolk, Tel Aviv, Israel). Hyperthyroidism was induced by daily injections of L-T3 as indicated, in 0.05 N NaOH in saline for 5 d. Control animals were injected with the vehicle only. Hypothyroidism was induced by 0.025% (wt/vol) methimazole in the drinking water for 4 wk. Serum T3 levels were measured when the animals were killed, 20 h after the last T3 injection, by RIA (kit from Diagnostic Products Corp., Los Angeles, CA) and were 0.6 ± 0.2, 3.9 ± 0.5, 3.5 ± 0.4 and 15 ± 3 nM in hypothyroid rats, euthyroid rats, and rats treated with 2 and with 50 μg T3/100 g body weight (BW), respectively. Fed animals from all groups were killed between 0800–0900 h by injection of 0.1 ml/100 g BW of a mixture containing 85 mg/ml ketamine and 3 mg/ml xylazine. The experimental protocol was approved by the ethical committee for animal and human experimentation of Hebrew University.
Mitochondria isolation
Rat liver mitochondria were isolated as previously described with slight modifications (31). Livers were homogenized in 300 mM sucrose, 20 mM Tris-HCl (pH 7.4), 2 mM EGTA, 0.1 μg/ml phenylmethylsulfonylfluoride, 1 μg/ml leupeptin, and 0.7 μg/ml pepstatin, followed by centrifugation at 1,100 x g for 10 min at 4 C. The supernatants were filtered through gauze and additionally centrifuged at 1,100 x g for 10 min at 4 C, followed by centrifugation at 10,800 x g for 10 min at 4 C for precipitating the crude mitochondrial fraction. Mitochondrial matrix was prepared as described previously (32).
Cells
Jurkat cells stably transfected with pEF-puro-Bcl2 expression plasmid or with the respective empty vector, as described by Huang et al. (33), were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 11 mM HEPES, 22 mM glutamine, 100 U penicillin/ml, and 0.1 mg streptomycin/ml. For T3 treatment, grown Jurkat cells were cultured in RPMI 1640 medium containing 10% charcoal-stripped FCS (Biological Industries, Beit Hemeek, Israel), followed by culture in RPMI 1640 medium containing 10–1000 nM T3 for the indicated time periods. One half volume of the culture medium was exchanged every other day. Where indicated, Cyclosporin A (4 μg/ml) was added to the culture 48 h before analysis.
Rat pituitary GH3 cells were grown in 30-mm culture dishes in MEM (Biological Industries) supplemented with 10% FCS, 0.1% glucose, and 20 mM glutamine. Cells were depleted of T3 using charcoal-stripped FCS. For TH treatment, 100–500 nM T3 and/or 7.5 μg/ml Cyclosporin A were added as indicated to the culture medium for 48 h before analysis.
HeLa and COS-7 cells were cultured in DMEM (Biological Industries) supplemented with 10% FCS, 50 U penicillin/ml, and 50 μg streptomycin/ml. Cells grown to 50% confluence on round glass coverslips, 12 mm in diameter, were transiently transfected using the calcium-phosphate precipitation with pcDNA3-green fluorescence protein (pcDNA3-GFP; 0.5 μg), pcDNA3-ANT2 (0.5–2.0 μg), or pcDNA3-Cyclophilin D (0.2–3.0 μg DNA) as indicated. After 6 h of incubation with the DNA precipitate, the cells were washed twice with PBS and cultured in fresh medium.
Mitochondrial membrane potential ()
Twenty-four-hour-transfected HeLa cells were incubated for 30 min in DMEM containing 150 nM chloromethyl-X-rosamine (MitoTracker red, Molecular Probes, Eugene, OR) at 37 C in the dark. Cells were washed with Hanks’ balanced salt solution and fixed in 3% paraformaldehyde for 10 min. The was determined by confocal microscopy (LSM 410, Carl Zeiss, Inc., New York, NY) by measuring the MitoTracker red intensity of GFP-transfected cells compared with neighboring, nontransfected cells, using ImagerPro Plus software (Media Cybernetics, Silver Spring, MD).
GH3 cells were incubated for 30 min in MEM containing 150 nM MitoTracker red at 37 C in the dark, followed by replacing the medium with Hanks’ balanced salt solution. The of living, nonfixed cells was immediately determined by confocal microscopy by measuring the MitoTracker red intensity of images of T3-treated cells compared with untreated cells.
Jurkat cells (1 x 106 cells) were incubated at 37 C in the dark for 30 min in RPMI 1640 medium containing 40 nM 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3); Molecular Probes] (34). Cells were centrifuged at 1000 x g for 2 min, and the pellet was washed once by resuspension in 400 μl PBS. The of washed cells was immediately analyzed by flow cytometric scanning (FACS; FL-2 setting; 20,000 events/analysis) using CellQuest software (BD Biosciences, Mountain View, CA). Uncoupling by carbonyl cyanide m-chlorophenylhydrazone (CCCP) was used as a positive control.
Cyclophilin D immunostaining
HeLa cells transfected with Cyclophilin D were fixed in 3% paraformaldehyde for 10 min and permeabilized by 0.1% Triton X-100 in a buffer containing 50 mM Tris-HCl (pH 7.6), 0.15 M NaCl, and 3% (wt/vol) BSA for 25 min. Permeabilized cells were washed and incubated with the same buffer for an additional 30 min for blocking nonspecific binding, followed by reacting the cells with rabbit antiCyclophilin D for 3 h and with secondary antirabbit Cy5 antibody for 1 h at room temperature.
Western blot analysis
Respective cells (1 x 107 to 3 x 107/sample) or liver tissue were homogenized by a hand-held glass homogenizer or by Polytron (Brinkmann Instruments, Westbury, NY), respectively, in 3 vol lysis buffer containing PBS, 1% Triton, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1 mM phenylmethylsulfonylfluoride, 10 μg leupeptin/ml, and 1 μg aprotinin/ml. Isolated mitochondria were suspended in lysis buffer as described above. After incubation in lysis buffer for 30 min at 4 C, respective lysates were cleared by centrifugation at 15,000 x g for 30 min, and supernatants were kept at –70 C. Equivalent amounts of proteins (80 μg) were resolved by 12.5% SDS-PAGE under reducing conditions and were transferred onto polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA) or cellulose nitrate membranes (Schleicher & Schuell, Dassel, Germany). Blots were probed with the indicated first antibody, followed by horseradish peroxidase-labeled second antibody. Bands were analyzed by ImageQuant software (Molecular Devices, Sunnyvale, CA).
Northern blot analysis
Total RNA was prepared from rat livers using the Ez-RNA extraction kit (Biological Industries). RNA (25 μg) was electrophoresed in a 1% formaldehyde agarose gel, blotted onto nylon membrane (GeneScreen Plus, NEN Life Science Products, Boston, MA), and cross-linked at 80 C for 2 h. Cyclophilin D mRNA was probed by Northern blot hybridization with 620 bp Cyclophilin D cDNA prepared by RT-PCR from rat total RNA using the sense 5'-CTAGGACAGCAGCAGGCAGC-3' and antisense 5'-TTGAGCAGACAGGCCTGGCT-3' primers (U68544 in GenEMBL cDNA bank). Blots were visualized and quantitated by phosphorimager densitometry (BAS 1000, FUJIX, Tokyo, Japan).
Materials
Jurkat cells (stably transfected with Bcl2 or with the empty vector) were obtained from G. Hacker (Technische Universitat, Munich, Germany). pcDNA3-Cyclophilin D and pcDNA3-ANT2 expression vectors were obtained from S. Grimm (Max Planck Institute for Biochemistry, Martinsried, Germany). Cyclosporin A was from Novartis Pharmaceuticals (East Hanover, NJ). Rabbit polyclonal antirat and antihuman Bcl2 antibody (SC-492, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), rabbit antirat and antihuman Cyclophilin D antibody (PA1–028, Affinity BioReagents, Golden, CO), rabbit antirat VDAC antibody (PA1–954, Affinity BioReagents), mouse monoclonal antirat and antihuman Bak antibodies (AM04, Oncogene Research Products, San Diego, CA) and rabbit antihuman Bax antibody (SC-493, Santa Cruz Biotechnology, Inc.) were commercially available. Rabbit antirat Bax antibody was obtained from Atan Gross (Weizmann Institute of Science, Rehovot, Israel). Horseradish peroxidase-labeled antirabbit and antimouse secondary antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA).
Data analysis
Statistical analysis was performed by one-way repeated measure ANOVA with Student-Newman-Keuls test. When only two groups were compared, significance was analyzed by paired t test.
Results
MTP gating by T3
MTP gating by T3 in Jurkat cells has been evaluated by measuring mitochondrial of cells cultured in the presence or absence of added T3 for up to 22 d. The was measured by flow cytometry, using the -sensitive dye DiOC6(3), in the absence and presence of added Cyclosporin A. As shown in Fig. 1A and in line with findings previously reported by Mihara et al. (34), most Jurkat cells (90%) were characterized by high fluorescence, reflecting their polarized inner . A minor population (10%) consisted of low fluorescent cells, reflecting their collapsed . Treatment with variable T3 concentrations (10–1000 nM) for 14–22 d resulted in a limited (13–17%), but significant, Cyclosporin A-sensitive mitochondrial depolarization of the major cell population (Fig. 1). Depolarization was already saturated by 10 nM T3, and an effect of lower T3 concentrations cannot be ruled out. Culturing of Jurkat cells with T3 for 7 d did not result in mitochondrial depolarization. Cyclosporin A-sensitive mitochondrial depolarization of Jurkat cells by T3 treatment was accompanied by a 33% increase in the minor low cell population (Figs. 1A and 9D; see below).
FIG. 1. LC-MTP gating induced by T3 in Jurkat cells. Jurkat cells were cultured for 14–22 d, as described in Materials and Methods, in the absence or presence of T3 or Cyclosporin A as indicated. Cultured cells were stained with DiOC6(3) and analyzed by FACS as described in Materials and Methods. A, Representative flow cytometric analysis of in untreated, T3-treated (500 nM), or T3 (500 nM) and Cyclosporin A (4.0 μg/ml)-treated cells. B, Histogram summarizing the effect of increasing concentrations of T3 (0–1000 nM) in the absence or presence of Cyclosporin A on the mean fluorescence intensity of the highly stained (channels, 2 x 102 to 3 x 103; A) cell population. The mean fluorescence of untreated cells is defined as 1.0. Values shown are the mean ± SE of three independent experiments performed in triplicate. *, Significant compared with untreated cells (P < 0.05).
FIG. 9. Bcl2 protection of T3-induced mitochondrial depolarization. Jurkat cells transfected with Bcl2 or with vector alone (WT) were grown for 14–22 d in the absence or presence of T3 as indicated. Cultured cells were stained with DiOC6(3) and analyzed by FACS as described in Materials and Methods. A, The Bcl2 protein content of nontransfected and Bcl2-transfected Jurkat cells determined by Western blot analysis as described in Materials and Methods. B, Representative flow cytometric analysis of of untreated and T3 (500 nM)-treated Jurkat cells, transfected with Bcl2 (right panel) or with vector alone (left panel). C, Histogram summarizing the mean fluorescence intensity of untreated and T3-treated Jurkat cells transfected with Bcl2 () or with vector alone () and sampled during d 14–22. Values shown are the mean ± SE of three independent experiments. *, Significant compared with the respective untreated cells (P < 0.05). D, Histogram summarizing the percentage of low cells of untreated and T3-treated (500–1000 nM; see Fig. 7) Jurkat cells transfected with Bcl2 () or with vector alone () and sampled during d 14–22. Values shown are the mean ± SE of three independent experiments. *, Significant compared with non-T3-treated, non-Bcl2-transfected cells (P < 0.05). #, Significant, by paired t test, compared with non-T3-treated, non-Bcl2-transfected cells (P < 0.05).
MTP gating by T3 has been verified as well in GH3 cells by confocal microscopy using MitoTracker red. As shown in Fig. 2, culturing GH3 cells with T3 for 48 h resulted in Cyclosporin A-sensitive, T3-dependent mitochondrial depolarization, amounting to a 28% decrease in . These results in Jurkat and GH3 cells complement our previous studies using Ca2+-loaded liver mitochondria derived from T3-treated rats, where T3 treatment induced a Cyclosporin A-sensitive, 70% decrease in mitochondrial pH and (26).
FIG. 2. LC-MTP gating induced by T3 in GH3 cells. GH3 cells were cultured for 48 h in the absence or presence of T3 and Cyclosporin A as indicated, followed by staining with MitoTracker red as described in Materials and Methods. A, Representative confocal microscopic images of untreated and T3-treated cells. B, Histogram summarizing the fluorescence intensity of MitoTracker red of untreated and T3-treated (500 nM) cells in the absence or presence of Cyclosporin A. Each column represents the mean fluorescence ± SE of 50–70 images collected from three independent experiments. *, Significant compared with untreated cells (P < 0.05). #, Significant compared with T3-treated cells (P < 0.05).
T3-induced MTP components
Cyclosporin A-sensitive MTP gating induced by T3 treatment in rat liver mitochondria (26, 28) or in cells (Figs. 1 and 2) was investigated in terms of the effects of T3 on the expression and function of MTP protein components.
ANT2.
ANT2 is essentially the only ANT isoform of rat liver mitochondria and the only isoform induced by T3 treatment (35). The role of ANT2 in MTP gating was evaluated by measuring Cyclosporin A-sensitive depolarization of the of HeLa cells overexpressing ANT2. Cells expressing ANT2 were identified by their cotransfected GFP. As shown in Fig. 3A, overexpression of ANT2 resulted in decreased , as verified by MitoTracker red images of transfected compared with neighboring nontransfected cells. The remained unaffected in GFP-transfected cells in the absence of transfected ANT2. The extent of depolarization induced by overexpressed ANT2 was dependent on the ANT2 transfection load. Thus, transfection of cells with low ANT2 (0.5 μg) did not affect , whereas higher ANT2 (>2.0 μg) induced an average 40% decrease in (Fig. 3B), indicating that T3-induced ANT2 could putatively account for T3-induced uncoupling. However, mitochondrial depolarization induced by overexpressed ANT2 was not affected by Cyclosporin A (Fig. 3B), indicating that Cyclosporin A-sensitive MTP gating induced by T3 is not mediated by T3-induced ANT2. Results similar to those derived by MitoTracker red imaging were verified by measuring mitochondrial depolarization by tetramethylrhodamine (TMRM) (not shown).
FIG. 3. T3-induced ANT2 in MTP gating. A, Representative confocal microscopic images of HeLa cells transfected with expression plasmids for GFP (0.5 μg; blue image) together with pcDNA3 (3.0 μg; upper panel) or pcDNA3-ANT2 (3.0 μg; lower panel) and stained with MitoTracker red (green image). Note the depolarization specifically induced by ANT2 in cells cotransfected with GFP and ANT2. B, Histogram summarizing the ratio between the fluorescence intensity of MitoTracker red of cells transfected with variable concentrations of pcDNA3-ANT2 and neighboring nontransfected cells. Where indicated, Cyclosporin A (CSA; 7.5 μg/ml) was added to the culture 24 h before MitoTracker red staining. The fluorescence intensity ratio of cells transfected with pcDNA3 is defined as 1.0. Values shown are the mean ± SE of 150 images collected from eight independent experiments. *, Significant compared with pcDNA3-transfected cells (P < 0.05).
VDAC.
The effect of T3 on liver mitochondrial VDAC levels was analyzed by measuring VDAC protein content in liver mitochondria isolated from untreated or T3-treated euthyroid or hypothyroid rats (Fig. 4). Mitochondrial VDAC levels remained essentially unaffected by T3, indicating that MTP gating induced by T3 treatment is not mediated by T3-induced VDAC.
FIG. 4. Effect of T3 on mitochondrial VDAC protein level. The mitochondrial VDAC protein contents of euthyroid (E), hyperthyroid (T), hypothyroid (H), and T3-treated hypothyroid (HT) rats were determined by Western blot analysis as described in Materials and Methods. The top panel illustrates a representative gel. Densitometric intensity of euthyroid VDAC is defined as 1.0. Values shown are the mean ± SE of four rats.
Cyclophilin D.
Cyclophilin D transcript and protein levels were assayed in livers of euthyroid, T3-treated, hypothyroid and T3-treated, hypothyroid rats. T3 treatment resulted in 2-fold increase in liver Cyclophilin D transcript (Fig. 5A) and protein (Fig. 5B) levels, whereas hypothyroidism resulted in a significant 2-fold decrease in liver Cyclophilin D protein, which was fully restored by T3 treatment (Fig. 5B). T3-induced Cyclophilin D was enzymatically functional, as verified by the increased peptidyl-prolyl cis-trans isomerase activity in T3-treated rat liver mitochondria (Fig. 5C).
FIG. 5. T3-induced Cyclophilin D. A, Cyclophilin D transcript relative to 28S rRNA of liver extracts from euthyroid (E) and T3-treated (T) rats determined by Northern blot analysis. The densitometric intensity of euthyroid Cyclophilin D mRNA is defined as 1.0. Values shown are the mean ± SE of four rats. *, Significant compared with euthyroid rats (P < 0.05). B, Cyclophilin D protein content of liver extracts derived from euthyroid (E), T3-treated (T), hypothyroid (H), and T3-treated hypothyroid (HT) rats determined by Western blot analysis. The densitometric intensity of euthyroid cells is defined as 1.0. The top panel illustrates a representative gel. Values shown are the mean ± SE of four rats. *, Significant compared with euthyroid rats (P < 0.05). #, Significant compared with hypothyroid rats (P < 0.05). C, The peptidyl-prolyl cis-trans isomerase (PPIase) activity of liver mitochondrial matrix prepared from euthyroid (E) and T3-treated (T) rats was assayed as described previously (37 ) in the absence or presence of added Cyclosporin A (CSA; 7.5 μg/ml). The initial rate of PPIase activity of euthyroid mitochondrial matrix is defined as 1.0. Values shown are the mean ± SE of seven rats. +, Average of two experiments differing by less than 10%. *, Significant compared with euthyroid rats (P < 0.05).
The effect of increased Cyclophilin D levels on MTP gating was evaluated by analyzing the of HeLa cells overexpressing Cyclophilin D. HeLa cells overexpressing Cyclophilin D were identified by cotransfection with GFP or by Cy5-labeled antiCyclophilin D antibody. The of cells transfected or not transfected with Cyclophilin D was verified by MitoTracker red intensity compared with neighboring nontransfected cells. Cyclophilin D overexpression could be expected to result in decreased and increased MTP sensitivity to oxidative stress (10). Surprisingly, however, MitoTracker red intensity was maintained or even increased in cells overexpressing Cyclophilin D (Fig. 6, A and B), indicating that the remained essentially unaffected or was even hyperpolarized by overexpressed Cyclophilin D. Mitochondrial colocalization of MitoTracker red and Cyclophilin D was verified by the yellow overlay image of mitochondrial MitoTracker red (Fig. 6A, green image) and transfected Cyclophilin D (Fig. 6A, red image). The intramitochondrial localization of transfected Cyclophilin D was verified by analyzing the processed/intramitochondrial (18-kDa) and unprocessed/extramitochondrial (21-kDa) Cyclophilin D proteins (36) in Cyclophilin D-transfected COS-7 cells. As shown in Fig. 6C, most of the transfected Cyclophilin D was in its processed form (18 kDa), indicating its intramitochondrial localization. Results similar to those derived by MitoTracker red imaging were verified by measuring mitochondrial depolarization with TMRM (not shown).
FIG. 6. T3-induced Cyclophilin D in MTP gating. A, Representative confocal microscopic images of HeLa cells transfected with expression plasmids for GFP (0.5 μg; blue image) together with pcDNA3 (3.0 μg; upper panel) or pcDNA3-Cyclophilin D (3.0 μg; lower panel). Transfected cells were stained with MitoTracker red (green image) and immunostained with antiCyclophilin D antibody, followed by Cy5-labeled secondary antibody (red image). Colocalization of mitochondrial MitoTracker red and Cy5-labeled Cyclophilin D results in a yellow image, indicating Cyclophilin D targeting to mitochondria. B, Histogram summarizing the fluorescence intensity of MitoTracker red of HeLa cells transfected with increasing pcDNA3-Cyclophilin D. The fluorescence intensity of cells transfected with pcDNA3 is defined as 1.0. Values shown are the mean ± SE of 150 images collected from eight independent experiments. *, Significant as compared with pcDNA3-transfected cells (P < 0.05). C, Western blots of pcDNA3-transfected (left lane) and pcDNA3-Cyclophilin D (6 μg)-transfected (right lane) COS-7 cells stained for Cyclophilin D.
The lack of effect of overexpressed Cyclophilin D on mitochondrial MTP gating was verified under conditions of oxidative stress induced by tert-butyl hydroperoxide. Tert-butyl hydroperoxide-induced mitochondrial depolarization in cells overexpressing Cyclophilin D was similar to that in control cells (not shown). These results may indicate that LC-MTP gating by T3 is not accounted for by T3-induced Cyclophilin D.
Bcl2 family components.
The family of Bax-Bcl2 proteins is grouped into two main subfamilies: proapoptotic proteins (e.g. Bax, Bak, and others) and antiapoptotic proteins (e.g. Bcl2), which promote or inhibit MTP gating, respectively (reviewed in Refs. 37 and 38). The effect of T3 on the expression of representative proteins of the two subfamilies was investigated in livers of euthyroid; T3-treated, hypothyroid; and T3-treated, hypothyroid rats as well as in Jurkat cells. Treatment of euthyroid or hypothyroid rats with T3 induced a dose-dependent increase in liver mitochondrial Bax (Fig. 7A) and resulted in 3-fold increase in liver mitochondrial Bak (Fig. 7B). In contrast to Bax and Bak, liver mitochondrial Bcl2 was increased by 20-fold in hypothyroid rats and was decreased by 35-fold in T3-treated, hypothyroid rats (Fig. 8A). The reciprocal changes in Bax and Bak contrasted with those in Bcl2 resulted in 5- and 7.5-fold respective increases in liver mitochondrial Bax/Bcl2 and Bak/Bcl2 ratios in T3-treated animals and 20- and 25-fold respective decreases in liver mitochondrial Bax/Bcl2 and Bak/Bcl2 ratios in hypothyroid rats. These results were corroborated in Jurkat cells, where T3 treatment for 14–22 d resulted in a dose-dependent 50% increase in cellular Bax (Fig. 7C) together with a 30% decrease in Bcl2 levels (Fig. 8B) (34) concomitant with their T3-induced, Cyclosporin A-sensitive mitochondrial depolarization (Fig. 1). No significant effect of T3 on Bax or Bcl2 content was observed in Jurkat cells incubated in the presence of T3 for 7 d, in line with the 14- to 22-d period required for their T3-induced, Cyclosporin A-sensitive mitochondrial depolarization (Fig. 1).
FIG. 7. T3-induced Bax and Bak. A, Mitochondrial Bax protein content of euthyroid (E), hyperthyroid (50 μg T3/l00 g BW; T), hypothyroid (H), and T3-treated hypothyroid (HT) rats. The densitometric intensity of euthyroid cells is defined as 1.0. Values shown are the mean ± SE of four rats. Inset, Histogram of liver mitochondrial Bax content of hypothyroid rats treated with increasing T3 doses as indicated. The densitometric intensity of hypothyroid cells is defined as 1.0. Values shown are the mean ± SE of four rats. The top panels illustrate representative gels. *, Significant compared with euthyroid rats (P < 0.05). #, Significant compared with hypothyroid rats (P < 0.05). B, Mitochondrial Bak protein content of euthyroid (E), hyperthyroid (50 μg T3/l00 g BW; T), hypothyroid (H), and T3-treated (50 μg T3/l00 g BW) hypothyroid (HT) rats. The top panel illustrates a representative gel. Densitometric intensity of euthyroid cells is defined as 1.0. Values shown are the mean ± SE of four rats in each treatment group. *, Significant compared with euthyroid rats (P < 0.05). #, Significant compared with hypothyroid rats (P < 0.05). C, The Bax protein content of untreated Jurkat cells and cells treated with T3 for 22 d as indicated. The top panel illustrates a representative gel of cells treated with increasing T3 concentrations as indicated. The densitometric intensity of untreated Jurkat cells is defined as 1.0. Values are the mean ± SE of three independent experiments performed in triplicate. Because the T3-induced decrease in is saturated by T3 concentrations higher than 10 nM (Fig. 1B), the results of two experiments using 500 nM T3 and one experiment using 1000 nM T3 were grouped for statistical analysis. *, Significant compared with untreated cells (P < 0.05).
FIG. 8. Suppression of Bcl2 by T3. A, Mitochondrial Bcl2 protein contents of euthyroid (E), hyperthyroid (50 μg T3/l00 g BW; T), hypothyroid (H), and T3-treated (50 μg T3/l00 g BW) hypothyroid (HT) rats were determined by Western blot analysis as described in Materials and Methods. The top panel illustrates a representative gel. The densitometric intensity of euthyroid Bcl2 is defined as 1.0. Values shown are the mean ± SE of four rats. *, Significant compared with euthyroid rats (P < 0.05). #, Significant compared with hypothyroid rats (P < 0.05). B, The Bcl2 protein content of untreated and 22-d T3-treated Jurkat cells determined by Western blot analysis as described in Materials and Methods. The top panel illustrates a representative gel of cells treated with increasing T3 concentrations as indicated. The densitometric intensity of untreated Jurkat Bcl2 is defined as 1.0. Values shown are the mean ± SE of three independent experiments performed in triplicate using 500-1000 nM T3 (see Fig. 7). *, Significant compared with untreated cells (P < 0.05).
The changes observed in liver mitochondrial content of Bax-Bcl2 proteins were only partly accounted for by respective changes in the total cellular content of the proteins concerned. Thus, the 2.9-fold increase in liver mitochondrial Bak (Fig. 7B) was correlated with a similar 3.0 ± 0.4-fold (P < 0.05) increase in total cellular Bak content, indicating that the T3-induced increase in mitochondrial Bak could be accounted for by its increased expression level. However, the 2.6-fold decrease in liver mitochondrial Bcl2 induced by T3 treatment (Fig. 8A) was in contrasted to the T3-induced 3.4 ± 0.7-fold (P < 0.05) increase in total cellular Bcl2 (not shown), indicating that suppression of liver mitochondrial Bcl2 by T3 treatment resulted from T3-induced factors other than its mere expression level.
The induction/suppression of Bax-Bcl2 family proteins by T3 in the context of MTP gating was evaluated in Jurkat cells overexpressing Bcl2 (Fig 9A). T3 treatment resulted in a Cyclosporin A-sensitive, 13–17% decrease in the of the major cell population (Fig. 1, A and B, and Fig. 9, B and C) accompanied by a 33% increase in the minor population of low cells (Fig. 9, B and D). However, overexpression of Bcl2 resulted in essentially eliminating T3-induced mitochondrial depolarization of the high cell population (Fig. 9, B and C) as well as eliminating the T3-induced increase in the low cell population (Fig. 9, B and D). Hence, suppression of mitochondrial Bcl2 combined with induction of mitochondrial Bax and Bak by T3 treatment may account for MTP gating by TH.
Discussion
TH-induced MTP components
Mitochondrial depolarization induced by T3 in Jurkat and GH3 cells, as reported here, conforms to LC-MTP gating. MTP involvement is indicated by its sensitivity to Cyclosporin A, whereas the limited extent of mitochondrial depolarization induced by T3 conforms to low conductance permeability transition. These findings complement those of our previous study, where T3 treatment in vivo combined with mitochondrial Ca2+ loading in vitro resulted in definitive Cyclosporin A-sensitive MTP gating, leading to extensive mitochondrial depolarization and mitochondrial swelling (26).
MTP gating induced by T3 was pursued in this study in terms of MTP components induced by T3 treatment, which could account for sensitization to MTP gating. T3 treatment is reported to result in pronounced increases in liver mitochondrial Bax and Bak together with a pronounced decrease in Bcl2, whereas hypothyroidism resulted in opposite effects that were reversed by T3. Also, the kinetics of T3-induced MTP gating in Jurkat cells was correlated with those of changes in their contents of respective Bax-Bcl2 proteins. Because the resultant effect of Bax-Bcl2 proteins on MTP gating is determined by the ratio between pro- and antiapoptotic proteins (39, 40), the effect of T3 is amplified by oppositely modulating pro- and antiapoptotic proteins of the Bax-Bcl2 family. Moreover, protection of T3-induced MTP gating by overexpressed Bcl2 may indicate that respective Bax-Bcl2 proteins induced/suppressed by T3 treatment transduce MTP gating induced by T3. MTP gating has indeed been repeatedly reported to be positively or negatively affected by proapoptotic (e.g. Bax and Bak) and antiapoptotic (e.g. Bcl2) proteins of the Bcl2 family, respectively (reviewed in Refs. 37 and 38). Bax-Bcl2 family proteins may directly interact with other MTP components such as ANT (41, 42) or VDAC (43), and when overexpressed or added to isolated mitochondria, they may specifically induce (e.g. Bax and Bak) (44, 45, 46) or antagonize (e.g. Bcl2) (47) MTP gating. Similarly, depletion of proapoptotic Bax or Bak results in failure of MTP gating (41, 47, 48), whereas Bcl2 inactivation results in definitive MTP gating triggered by oxidative stress (49).
The mode of action of T3 in enriching/depleting mitochondrial members of the Bax-Bcl2 family remains to be investigated in terms of the expression of nuclear and mitochondrial genes putatively involved in translocating and anchoring Bax-Bcl2 proteins to the MTP complex of specific cell types. The T3-induced increase in total liver Bcl2, in contrast with its mitochondrial depletion (see Results), may indicate that T3-induced factors other than expression levels of Bax-Bcl2 proteins (e.g. their phosphorylation and mitochondrial translocation) are involved in modulating the liver mitochondrial content of respective MTP components.
In contrast to proteins of the Bax-Bcl2 family, TH-induced MTP gating does not seem to be transduced by TH-induced ANT2 or Cyclophilin D. In light of its structural (10, 13) and/or regulatory (14) functions in the MTP complex, ANT2 expression induced by T3 treatment (35) could, in principle, account for liver MTP gating induced by T3. Overexpression of mitochondrial ANT2 was indeed found here to result in extensive mitochondrial depolarization, similar to that previously reported for overexpressed ANT1 (50, 51, 52). The discrepancy between our findings and the previously reported lack of effect of overexpressed ANT2 on mitochondrial depolarization and apoptosis (50, 52) could result from differences in ANT2 transfection yields or its mitochondrial targeting. However, mitochondrial depolarization induced by overexpressed ANT2 was insensitive to Cyclosporin A (Fig. 3B), reflecting the formation of an MTP-nonrelated ANT channel (53) or of Cyclosporin A-insensitive MTP (21) by overexpressed ANT2. The lack of an obligatory linkage between MTP and ANT conforms to recent findings pointing to MTP gating by proapoptotic ligands in liver cells and isolated mitochondria lacking ANT altogether (14). These findings taken together with the lack of effect of T3 treatment on VDAC levels (Fig. 4) imply that T3-induced, Cyclosporin A-sensitive MTP gating is not accounted for by modulating the mitochondrial content of ANT or VDAC.
Induction of Cyclophilin D by T3 treatment could similarly be expected to result in Cyclosporin A-sensitive MTP gating in light of the putative role of Cyclophilin D in modulating ANT affinity for Ca2+, which is aborted by Cyclosporin A (10, 12, 13). In vivo T3 treatment was indeed found here to induce Cyclophilin D transcript, protein, and peptidyl-prolyl cis-trans isomerase activities. Surprisingly, however, overexpression of Cyclophilin D and its mitochondrial targeting resulted in mitochondrial hyperpolarization, rather than MTP gating. Our results do not agree with those previously reported by De Giorgy et al. (54), but corroborate those presented by Lin et al. (55), who reported that was hyperpolarized in cells overexpressing Cyclophilin D. Furthermore, our results are in line with previous reports that overexpressed Cyclophilin D desensitized cells to apoptotic stimuli or to protect cells from mitochondrial depolarization and apoptosis induced by overexpression of ANT1 (50, 51, 55). Hence, induction of Cyclophilin D by T3 may not account for T3-induced MTP gating.
MTP gating by TH
Our previously (26, 27) and presently reported results may indicate that in vivo TH treatment results in low conductance MTP or definitive MTP gating, depending on a variety of physiological or pathological signals. The two MTP modes have distinct characteristics and may serve distinct functions. Thus, TH-induced LC-MTP gating represents an innocuous limited mitochondrial depolarization (23, 24, 25) and is proposed to control metabolic efficiency by modulating the extent of coupling of mitochondrial oxidative phosphorylation. TH has indeed long been realized to increase mitochondrial mass and oxygen consumption together with decreasing the mitochondrial phosphate potential and mitochondrial redox state (56, 57). Mitochondrial uncoupling by TH is exerted at the level of the mitochondria, as verified by the stimulation of state 4 respiration, increase in proton leak, and decrease in membrane potential of mitochondria isolated from TH-treated rats (58, 59). Because mitochondrial uncoupling accounts for 25% and 50% of state 4 respiration in liver and muscle, respectively, and the phosphorylating machinery competes with mitochondrial proton leaks for the same driving force, the overall enthalpy required for carrying out a given task may be dominated by TH-induced LC-MTP gating under both basal (57) as well as working (59) conditions.
In contrast to LC-MTP gating, TH-induced definitive MTP gating results in extensive mitochondrial depolarization and swelling mediated by synchronized, irreversible MTP opening, leading to apoptosis (37, 38). TH-induced apoptosis of peripheral blood T lymphocytes has indeed been exemplified in vivo in Graves’ disease patients (34). The definitive MTP gating mode may serve the role of organ development, exemplified by TH-induced tadpole tail apoptosis in the course of amphibian metamorphosis (60, 61), or, alternatively, may pose a risk under conditions of cardiac ischemia-reperfusion damage (62, 63).
TH treatment is proposed to promote the LC and definitive gating modes of MTP by enriching/depleting mitochondrial Bax-Bcl2 proteins. Changes in mitochondrial Bax-Bcl2 proteins may affect spontaneous MTP gating frequency, gating synchronization of individual pores, or sensitivity to Ca2+ or oxidative stress. The increase in the number of low Jurkat cells induced by T3 concomitantly with LC-MTP gating of the major cell population (Fig. 1) and the abrogation of both by overexpressed Bcl2 (Fig. 9) indicate that LC-MTP gating induced by T3 may promote drift into definitive MTP gating (Fig 10). Hence, depending on the prevailing triggering signals, TH-sensitized MTP may either be operating in its restrained physiological constraints in the context of LC-MTP gating or drift nonreversibly into definitive MTP gating.
FIG. 10. MTP gating by T3. The T3-induced increase in the Bax/Bcl2 ratio is proposed to drive MTP gating from its closed hypothyroid configuration to its low conductance hyperthyroid configuration, resulting in limited depolarization and progressive decoupling of mitochondrial oxidative phosphorylation with concomitant increase in calorigenesis. Exposure of T3-sensitized MTP to oxidative stress or increased mitochondrial Ca2+ may result in definitive MTP gating, leading to collapsed and apoptosis.
Acknowledgments
The kind generosity of G. Hacker (Jurkat cells), S. Grimm (pcDNA-ANT2 and Cyclophilin D expression vectors), A. Gross (rabbit antirat Bax antibody), and Novartis Pharmaceuticals (Cyclosporin A) is deeply acknowledged.
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