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Am J Physiol Renal Physiol 294: F499-F507, 2008. First published December 26, 2007; doi:10.1152/ajprenal.00415.2007
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Effects of targeted Bcl-2 expression in mitochondria or endoplasmic reticulum on renal tubular cell apoptosis

Kirti Bhatt,1,* Leping Feng,1,* Navjotsingh Pabla,1 Kebin Liu,2 Sylvia Smith,1 and Zheng Dong1

Departments of 1Cellular Biology and Anatomy and of 2Biochemistry, Medical College of Georgia and Veterans Affairs Medical Center, Augusta, Georgia

Submitted 6 September 2007 ; accepted in final form 21 December 2007


   ABSTRACT
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MATERIALS AND METHODS
 RESULTS AND DISCUSSION
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Bcl-2 family proteins are central regulators of apoptosis. As the prototypic member, Bcl-2 protects various types of cells against apoptotic insults. In mammalian cells, Bcl-2 has a dual subcellular localization, in mitochondria and endoplasmic reticulum (ER). The respective roles played by mitochondrial and ER-localized Bcl-2 in apoptotic inhibition are unclear. Using Bcl-2 constructs for targeted subcellular expression, we have now determined the contributions of mitochondrial and ER-localized Bcl-2 to the antiapoptotic effects of Bcl-2 in renal tubular cells. Wild-type Bcl-2, when expressed in renal proximal tubular cells, showed partial colocalizations with both cytochrome c and disulfide isomerase, indicating dual localizations of Bcl-2 in mitochondria and ER. In contrast, Bcl-2 constructs with mitochondria-targeting or ER-targeting sequences led to relatively restricted Bcl-2 expression in mitochondria and ER, respectively. Expression of wild-type and mitochondrial Bcl-2 showed significant inhibitory effects on tubular cell apoptosis that was induced by cisplatin or ATP depletion; however, ER-Bcl-2 was much less effective. During ATP depletion, cytochrome c was released from mitochondria into the cytosol. This release was suppressed by wild-type and mitochondrial Bcl-2, but not by ER-Bcl-2. Consistently, wild-type and mitochondrial Bcl-2, but not ER-Bcl-2, blocked Bax activation during ATP depletion, a critical event for mitochondrial outer membrane permeabilization and cytochrome c release. In contrast, ER-Bcl-2 protected against apoptosis during tunicamycin-induced ER stress. Collectively, the results suggest that the cytoprotective effects of Bcl-2 in different renal injury models are largely determined by its subcellular localizations.

Bcl-2; cisplatin; adenosine 5'-triphosphate depletion; apoptosis; mitochondria; endoplasmic reticulum

BCL-2 FAMILY PROTEINS are critical regulators of apoptosis in a variety of cells from Caenorhabditis elegans to mammals (7, 8, 14, 18, 19, 30). Characterized by the presence of Bcl-2 homology (BH) domains, these proteins can be proapoptotic or antiapoptotic. Antiapoptotic members, like Bcl-2 and Bcl-XL, contain four BH domains, whereas proapoptotic members such as Bax and Bak have three BH domains, and others have only one BH domain, the BH3. It is generally accepted that the balance between proapoptotic and antiapoptotic Bcl-2 family proteins determines the fate of a cell under stress, to survive or to die by apoptosis (7, 8, 14, 18, 19, 30).

In models of ischemic kidney injury, an important role of Bcl-2 family proteins has been demonstrated in apoptosis of renal tubular cells (3, 9, 24). Specifically, mitochondrial outer membrane is permeabilized in these cells during hypoxia and ATP depletion, followed by cytochrome c release, caspase activation, and apoptosis (11, 28). Activation of Bax and Bak, two proapoptotic Bcl-2 family proteins, is a key to mitochondrial damage under these conditions. Notably, in humans, mitochondrial damage by Bax and Bak seems to be critical to apoptotic cell death in ischemically injured kidneys (6, 34).

Similarly, Bcl-2 family proteins appear to regulate tubular cell apoptosis during cisplatin nephrotoxicity. In cultured LLC-PK1 proximal tubular cells, cisplatin induces Bax activation, accompanied by cytochrome c release and the activation of caspase-9 (25). Our recent work further suggests that p53 is activated by cisplatin in tubular cells and induces p53-upregulated modulator of apoptosis (PUMA-{alpha}), a proapoptotic BH3-only protein (16). PUMA-{alpha} then targets mitochondria, where it interacts and antagonizes Bcl-XL, leading to Bax/Bak activation and outer membrane permeabilization (16). These observations are further supported by our latest work showing that cisplatin-induced nephrotoxicity is ameliorated in Bax-deficient mice (32). Of note, in both ischemic and cisplatin nephrotoxic injury, tubular cell apoptosis can be inhibited by Bcl-2 and its homolog Bcl-XL (11, 28, 31).

Bcl-2 is the prototypic member of the Bcl-2 family. Although the cytoprotective effects of Bcl-2 have been demonstrated in a variety of apoptotic models, the exact mechanism is not entirely clear. It is generally believed that Bcl-2 can sequester the proapoptotic members and thus neutralize their apoptotic activity (7, 8, 14, 18, 19, 30). In addition, recent work suggests that Bcl-2 may also regulate Ca2+ homeostasis in endoplasmic reticulum (ER) to modulate the cellular sensitivity to apoptosis (21, 26). Bcl-2 has dual subcellular localizations, mitochondria and ER. It is not entirely clear how the subcellular localization would affect the antiapoptotic efficacy of Bcl-2. The current study was designed to address this question using Bcl-2 constructs with targeted expression in mitochondria or ER.


   MATERIALS AND METHODS
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Materials. The rat kidney proximal tubular cell line (RPTC) was originally obtained from Dr. U. Hopfer (Case Western Reserve University, Cleveland, OH) and maintained for experiment as previously described (11, 15, 16, 28). The stable Bcl-2-overexpressing RPTC cell line was generated and used in our previous study (17, 28). Antibodies were purchased from the following sources: monoclonal anticytochrome c from BD Biosciences (San Diego, CA), polyclonal antiactive Bax from Upstate Biotechnology (Lake Placid, NY), polyclonal anti-Bcl-2 from Santa Cruz (Santa Cruz, CA), monoclonal antidisulfide isomerase (PDI) from Abcam (Cambridge, MA), and fluorescein isothiocycnate (FITC)- or Cy3-conjugated secondary antibodies from Pierce Biotechnology (Rockford, IL). Other reagents, including azide and cisplatin, were purchased from Sigma (St. Louis, MO).

Bcl-2-expressing plasmids. Wild-type Bcl-2 and the Bcl-2 constructs containing either the ER or the outer mitochondrial membrane insertion sequences were kindly provided by Dr. David W. Andrews at McMaster University [Hamilton, Ontario, Canada (36)]. The Bcl-2 sequences were released from the original plasmids with Hind III, or ApaI and Hind III, and subcloned into pcDNA3.1/Hygro (Invitrogen) for transfection. The plasmids were used in our recent study (5).

Transient transfection of RPTC. Transient transfection was conducted using Lipofectamine 2000 (Invitrogen) as described previously (35). Briefly, cells were plated on at 0.3–0.75 x 106/35-mm dish to reach 50–60% confluence after overnight growth. The cells were then transfected with 1.0 µg of a Bcl-2 plasmid or the empty vector pcDNA3.1 (Invitrogen). pEGFP-C3 (0.25 µg/dish; Clonetech) was cotransfected to label the transfected cells with green fluorescent protein. The transfected cells were used in the next day for experimental treatment.

Induction of apoptosis. Apoptosis was induced in RPTC by ATP depletion and cisplatin treatment as described previously (11, 17). For ATP depletion, the cells were incubated with 10 mM azide for 3 h in glucose-free Krebs-Ringer bicarbonate solution. Subsequently, groups of cells were returned back to full culture medium for recovery. ATP depletion during azide treatment and recovery of ATP levels after returning to full culture medium were shown previously (12). In this experimental model, mitochondrial events of apoptosis (i.e., cytochrome c release and Bax translocation) occur during ATP depletion, and apoptotic morphology develops during the recovery period. For cisplatin treatment, the cells were incubated with 20 µM cisplatin in culture medium for the indicated time.

Examination of apoptosis. Cells, after various treatments, were fixed with 4% paraformaldehyde and incubated with 10 µg/ml of Hoechst 33342 for 2–5 min to stain the nuclei. The cells were then examined by fluorescence microscopy. The morphological examination was focused on the green fluorescent protein (GFP)-labeled cells, which were transfected with different Bcl-2 constructs or the control empty vector. Typical apoptotic morphology was indicated by cellular shrinkage, formation of apoptotic bodies or blebs, and nuclear condensation and fragmentation. For quantification, ~100 transfected cells were identified in each dish by GFP fluorescence. Morphology of these cells was evaluated carefully to determine the percentage of apoptosis. Usually, duplicate dishes were used for each condition in an experiment. The experiments were repeated for at least four times for statistical analysis. To validate the morphological analysis, caspase activation was examined by analyzing the immunofluorescence of active caspase-3 as described in our previous studies (11, 17). Briefly, cells were fixed and exposed to an antibody that specifically recognized the active fragment of caspase-3. The cells were then incubated with the Cy3-conjugated secondary antibody for examination by fluorescence microscopy. The examination was focused on the GFP-labeled transfected cells.

Immunofluorescence. Indirect immunofluorescence was conducted as described previously (13, 28, 32). Briefly, RPTC were grown on collagen-coated glass cover slips. For immunofluorescence of cytochrome c, cells were fixed with a modified Zamboni's fixative containing picric acid and 4% paraformaldehyde and then blocked with 2% normal goat serum. Finally, the cells were incubated with a mouse monoclonal anticytochrome c antibody, followed by exposure to Cy3-labeled goat antimouse secondary antibody. For immunofluorescence of active Bax, the cells were fixed with 4% paraformaldehyde, blocked with 2% normal goat serum, exposed to active Bax antibody, and finally incubated with a Cy3-labeled secondary antibody. For double immunofluorescence of Bcl-2 and cytochrome c or PDI, the cells following fixation were exposed to a mixture of primary antibodies (rabbit anti-Bcl-2 and mouse anticytochrome c or PDI), followed by secondary antibodies (FITC-labeled goat antirabbit and Cy3-labeled goat antimouse). Immunofluorescence was examined by confocal microscopy using imaging software (LSM 510 Meta; Carl Zeiss).

Statistical analysis. Data were expressed as means ± SD (n ≥ 3). Statistical differences were determined by Dunnett's multiple-comparison posttest following ANOVA using the GraphPad Prism software; P < 0.05 was considered significantly different.


   RESULTS AND DISCUSSION
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 MATERIALS AND METHODS
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Subcellular localization of wild-type Bcl-2 expressed in RPTC. Bcl-2 has a stretch of hydrophobic amino acids at the carboxyl terminus, which is recognized as the transmembrane domain to direct Bcl-2 localization to intracellular membranes, particularly the ER and the outer mitochondrial membrane. Deletion of the transmembrane sequence prevents Bcl-2 insertion in membranes and diminishes its antiapoptotic activity (22), indicating an essential role for membrane targeting in Bcl-2 function. In RPTC used in our study, Bcl-2 expression was low, making it difficult to examine its subcellular localization. We therefore used the RPTC that were stably transfected with wild-type Bcl-2 (28). In one set of dishes, double immunofluorescence was examined for Bcl-2 and cytochrome c, a protein normally restricted in mitochondria. Another set of dishes was examined for double immunofluorescence of Bcl-2 and PDI, an ER marker protein. As shown in Fig. 1, both cytochrome c and PDI staining was perinuclear, but cytochrome c staining was more punctate, which is consistent with the general structure and organization of mitochondria and ER. Bcl-2 in these cells showed a cytoplasmic staining with some apparent structural or organellar signals at the perinuclear region. Superimposing of the images indicated that some Bcl-2 colocalized with cytochrome c in mitochondria (Fig. 1A), whereas some Bcl-2 colocalized with PDI in ER. The results suggest that Bcl-2 has a dual subcellular localization in proximal tubular cells, mitochondrion and ER.


Figure 1
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  		Fig. 1. Dual localization of expressed Bcl-2 in mitochondria and endoplasmic reticulum (ER) in rat kidney proximal tubular cells (RPTC). RPTC stably transfected with Bcl-2 were fixed for double immunofluorescence analysis of Bcl-2 and cytochrome c (Cyt.c) or disulfide isomerase (PDI). The same cells were examined by confocal fluorescence microscopy for Bcl-2 (green) and cytochrome c (red) or PDI (red). Images of the same cells were superimposed to reveal their colocalization.

 
Targeted Bcl-2 expression in mitochondria or ER in RPTC. By replacing the carboxyl-terminal transmembrane domain in Bcl-2 with mitochondrion or ER-targeting sequences, two plasmids for targeted Bcl-2 expression in mitochondria or ER were constructed (36). The sequences were subcloned into pcDNA3.1/Hygro for transfection into RPTC. To determine the subcellular localization of transfected Bcl-2, double immunofluorescence was examined for Bcl-2 and cytochrome c or PDI. Shown in Fig. 2A is a cell transfected with Bcl-2 containing a mitochondrion-targeting sequence; Bcl-2 immunofluorescence showed a perinuclear filamentous staining with a typical mitochondrial morphology. Consistently, the Bcl-2 staining overlapped with that of cytochrome c in the same cell. In contrast, the cells transfected with Bcl-2 containing an ER-targeting sequence showed a much finer Bcl-2 staining in cytoplasm (Fig. 2B) that largely overlapped with PDI staining (Fig. 2B). These cells also had some Bcl-2 staining in subcellular locations other than ER, probably because of Bcl-2 overexpression and/or the staining of endogenous Bcl-2. Of note, in both dishes (transfected with mitochondrial or ER Bcl-2), there were cells that were not transfected, and, as a result, these cells showed a weak fluorescence staining. Together, these experiments showed the subcellular expression specificity of the Bcl-2 constructs in RPTC that were used in subsequent studies.


Figure 2
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  		Fig. 2. Targeted Bcl-2 expression in mitochondria or ER in RPTC. RPTC were transfected with Bcl-2 constructs containing either a mitochondrion or ER-targeting sequence. The cells were then fixed for immunofluorescence of Bcl-2 (green) and cytochrome c (red) or PDI (red). Images of the same cells were superimposed to reveal the colocalization of Bcl-2 with cytochrome c or PDI. Solid line, Bcl-2-transfected cells; dashed lines, untransfected cells.

 
Effects of targeted Bcl-2 expression on cisplatin-induced RPTC apoptosis. Cisplatin is a widely used chemotherapy drug with major side effects in kidneys, inducing tubular cell death and acute renal failure (2, 23). In vitro studies using cultured LLC-PK1 cells have suggested the involvement of the intrinsic mitochondrial pathway in tubular cell apoptosis during cisplatin treatment (25). This inference is supported by our latest in vivo study showing that Bax is activated by cisplatin in C57BL/6 mice and cisplatin nephrotoxicity is ameliorated in Bax-deficient animals (32). In RPTC, overexpression of wild-type Bcl-2 blocks Bax activation, cytochrome c release from mitochondria, caspase activation, and subsequent apoptosis (17). However, it is unknown whether the antiapoptotic effects of Bcl-2 depend on its subcellular localization. To address this question, wild-type Bcl-2 (wBcl-2), mitochondrion-targeting Bcl-2 (mBcl-2), and ER-targeting Bcl-2 (eBcl-2) were separately transfected into RPTC. To identify the transfected cells, GFP was cotransfected with each of the Bcl-2 plasmids to label the successfully transfected cells. After transfection, the cells were incubated with 20 µM cisplatin for 16 h, and apoptosis was examined by cellular and nuclear morphology revealed by Hoechst staining. Representative morphology of wBcl-2 or empty vector transfected cells is shown inFig. 3, A and B. Without treatment, control cells showed a healthy cellular and nuclear morphology, regardless the transfection with Bcl-2 or empty vector (Fig. 3, A and B). After cisplatin treatment, many cells in the empty vector transfection group assumed a typical apoptotic morphology, characterized by cellular and nuclear condensation and fragmentation (Fig. 3A). In contrast, the majority of wBcl-2 transfected cells maintained a healthy nonapoptotic morphology (Fig. 3B). For quantification, the percentage of apoptotic cells in the transfected (GFP-labeled) population was determined by cell counting. As shown in Fig. 3C, control cells without cisplatin treatment showed a basal level of apoptosis. Cisplatin induced apoptosis in the cells transfected with the empty vector in a treatment time-dependent manner, reaching 72 ± 6% at 48 h. Cisplatin-induced apoptosis was reduced significantly by transfection with wBcl-2 (35 ± 3%) or mBcl-2 (39 ± 4%), whereas transfection with eBcl-2 was less effective (59 ± 4%). We further examined caspase activation by immunofluorescence using an antibody that specifically recognized the active fragment of caspase-3 (11, 17). After 24 h of cisplatin treatment, active caspase-3 was detected in 52% cells, which was significantly reduced by wBcl-2 (22 ± 3%) and mBcl-2 (21 ± 4%), but not by eBcl-2 (45 ± 6%). These results further suggested that mitochondrially localized Bcl-2 was more effective than ER-targeted Bcl-2 in cytoprotection against cisplatin injury. In addition, the active caspase-3 staining verified the morphological analysis of apoptosis, which was used for subsequent experiments in this study. To determine the expression levels of various Bcl-2 constructs, we collected whole cell lysates for immunoblot analysis (Fig. 3D). The results showed similar levels of expression for eBcl-2 and mBcl-2, whereas wBcl-2 expression was relatively lower. Of note, the sizes of the expressed Bcl-2 proteins were different (Fig. 3D) because eBcl-2 and mBcl-2 had ER and mitochondrial targeting sequences, respectively, and wBcl-2 did not. The immunoblot results indicate that the observed protective effects of mBcl-2 and wBcl-2 in these experiments were not because of their higher expression.


Figure 3
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  		Fig. 3. Effects of targeted Bcl-2 expression on cisplatin-induced RPTC apoptosis. RPTC were transfected with a Bcl-2 plasmid or empty vector. Green fluorescent protein (GFP) was cotransfected to label the transfected cells. After transfection, the cells were incubated with 20 µM cisplatin for 16 h and then stained with Hoechst 33342. A and B: representative images of cells transfected with empty vector (A) or wild-type Bcl-2 (B) with or without cisplatin treatment. C: cisplatin induced apoptosis in cells transfected with empty vector or various Bcl-2 plasmids (wBcl-2, mBcl-2, and eBcl-2 indicate wild-type, mitochondria-targeting, and ER-targeting Bcl2). Data are presented as means (n = 4); error bars are omitted for clarity. D: expression levels of wBcl-2, mBcl-2, and eBcl-2 following transfection in RPTC. Following transfection, whole cell lysates were collected for immunoblot of Bcl-2. The blots were reprobed for β-actin as a control.

 
Effects of targeted Bcl-2 expression on RPTC apoptosis induced by ATP depletion. The experiments described above showed that mitochondrial Bcl-2 was more effective than ER Bcl-2 in protecting against cisplatin-induced apoptosis in RPTC. We further extended the observation in a model of ATP depletion. ATP depletion is a determining factor of tubular cell injury and death during renal ischemia-reperfusion. Using RPTC, our previous work has suggested that apoptosis following ATP depletion involves Bax activation and cytochrome c release from mitochondria, which can be inhibited by overexpression of wild-type Bcl-2 (28). To determine the effects of targeted Bcl-2 expression, RPTC were cotransfected with GFP and one Bcl-2 plasmid. The transfected cells were then incubated with azide in glucose-free medium for 3 h, followed by recovery in full culture medium. Within 1 h of recovery, 42 ± 1% cells that were transfected with empty vector developed apoptotic morphology (Fig. 4, morphology not shown). Transfection with wBcl-2 or mBcl-2 suppressed apoptosis to 23 ± 2%. In contrast, transfection with eBcl-2 did not have significant inhibitory effects (Fig. 4).


Figure 4
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  		Fig. 4. Effects of targeted Bcl-2 expression on RPTC apoptosis following ATP depletion. RPTC were cotransfected with GFP and a Bcl-2 plasmid or empty vector. After transfection, the cells were subjected to 3 h of ATP depletion with 10 mM azide in glucose-free buffer, followed by 1 h recovery in full culture medium. The cells were then stained with Hoechst 33342 to examine apoptosis by morphological criteria in transfected (GFP-labeled) cells. Data are presented as means ± SD (n = 4). *Statistically significant difference from control. #Statistically significant difference from the treated group transfected with empty vector.

 
Effects of targeted Bcl-2 expression on RPTC apoptosis during tunicamycin-induced ER stress. One pathway of apoptosis is via ER stress (4). Although apoptosis triggered by ER stress may also involve mitochondrial events, the initial trigger is the stress in ER. The information about ER stress in renal pathophysiology is very limited. We reasoned that ER-targeting Bcl-2 might be more effective than mitochondrial Bcl-2 in protecting against ER stress-associated apoptosis in renal tubular cells. To test this possibility, RPTC were treated with tunicamycin, a commonly used inducer of ER stress. As shown in Fig. 5, tunicamycin induced 44 ± 9% apoptosis in vector-transfected cells, which was suppressed by eBcl-2 to 16 ± 4%. In contrast, mBcl-2 only had marginal protective effects. wBcl-2 also showed relatively low protective effects that may be related to the low wBcl-2 expression after transfection in RPTC (Fig. 3D). Recent work by Puthalakath et al. (27) has demonstrated a critical role for Bim in apoptosis triggered by ER stress. Consistently, we showed that Bim was induced by tunicamycin in RPTC (Fig. 5B). Together, the results suggest that the antiapoptotic action of Bcl-2 depends on its subcellular localization and the specific apoptotic pathways.


Figure 5
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  		Fig. 5. Effects of targeted Bcl-2 expression on tunicamycin-induced apoptosis in RPTC. A: RPTC were cotransfected with GFP and a Bcl-2 plasmid or empty vector. After transfection, the cells were treated with 1 µg/ml tunicamycin for 18 h. The transfected cells (GFP positive) were evaluated for their nuclear and cellular morphology to determine the %apoptosis. Data are presented as means ± SD (n = 3). *Statistically significant difference from control. #Statistically significant difference from the treated group transfected with empty vector. B: Bim induction by tunicamycin in RPTC. RPTC were treated with 1 µg/ml tunicamycin for indicated time to collect whole cell lysate for immunoblot analysis of Bim and β-actin.

 
Effects of targeted Bcl-2 expression on cytochrome c release during ATP depletion. A critical apoptotic event at the mitochondria is the release of apoptogenic factors or proteins, including cytochrome c. To confirm that mitochondrial Bcl-2 suppressed tubular cell apoptosis by blocking mitochondrial leakage of cytochrome c, we used the azide-induced ATP depletion model. This model was chosen mainly because cytochrome c was released from mitochondria during the period of ATP depletion when cells had yet to develop apoptosis (which required recovery in full culture medium); examination of cytochrome c release during ATP depletion would therefore avoid many of the secondary apoptotic effects (10, 28). As shown in Fig. 6, control cells had cytochrome c in mitochondria, showing a perinuclear organellar staining. After 3 h of ATP depletion with azide, the cells transfected with empty vector released cytochrome c, resulting a cytosolic staining (Fig. 6A). In contrast, the cells transfected with wBcl-2 maintained cytochrome c in mitochondria (Fig. 6B). Quantification by cell counting showed that 38% of the empty vector-transfected cells released cytochrome c during ATP depletion. Transfection with wBcl-2 and mBcl-2, respectively, reduced the percentage to 17 and 21%, whereas cytochrome c release was reduced slightly by eBcl-2 to 32% (Fig. 6C).


Figure 6
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  		Fig. 6. Effects of targeted Bcl-2 expression on cytochrome c release during ATP depletion. RPTC were cotransfected with GFP and a Bcl-2 plasmid or empty vector. After transfection, the cells were subjected to 3 h of ATP depletion with 10 mM azide in glucose-free buffer. The cells were then fixed for analysis of cytochrome c immunofluorescence. The distribution of cytochrome c in transfected (GFP-labeled) cells was examined by confocal microscopy. A and B: representative images of cells transfected with empty vector (A) or wild-type Bcl-2 (B) with or without azide treatment. C: percentage of cells with indicated plasmid transfection that released cytochrome c during azide-induced ATP depletion. Data are presented as means ± SD (n = 4). *Statistically significant difference from the treated group transfected with empty vector.

 
Effects of targeted Bcl-2 expression on Bax activation during ATP depletion. In the ATP depletion model, we showed previously that the activation of Bax and Bak, two multidomain proapoptotic Bcl-2 proteins, is critical to permeabilization of the outer mitochondrial membrane and release of cytochrome c (11, 20, 28). Upregulation of Bcl-2 can prevent Bax and Bak activation and, as a result, preserve the integrity of mitochondria and maintain cytochrome c in the organelles (11, 20, 28). We hypothesized that mitochondrial Bcl-2 would be more effective than ER Bcl-2 in blocking Bax activation during ATP depletion. To test this possibility, we examined Bax activation using an antibody that specifically recognizes active Bax. The antibody has been used successfully to detect Bax activation in situ in renal tissues (6, 33). RPTC were cotransfected with GFP and a Bcl-2 plasmid or empty vector and then subjected to azide-induced ATP depletion for 3 h. Shown in Fig. 7A are cells transfected with empty vector. Without azide treatment, there was weak background staining of active Bax (Fig. 7A). After azide treatment, many cells showed strong active Bax staining, which was perinuclear and appeared punctate (Fig. 7A). In the cells transfected with wild-type Bcl-2, Bax activation was ameliorated (Fig. 7B). The results of cell counting are summarized in Fig. 7C. Azide treatment for 3 h induced active Bax in 51% of the cells that were transfected with empty vector, 21% of the cells transfected with wBcl-2, 24% of the cells transfected with mBcl-2, and 50% of the cells transfected with eBcl-2.


Figure 7
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  		Fig. 7. Effects of targeted Bcl-2 expression on Bax activation during ATP depletion. RPTC were cotransfected with GFP and a Bcl-2 plasmid or empty vector. After transfection, the cells were subjected to 3 h of ATP depletion with 10 mM azide in glucose-free buffer. The cells were then fixed for immunofluorescence analysis of active Bax. The fluorescence signals were examined in transfected (GFP-labeled) cells by confocal microscopy. A and B: representative images of cells transfected with empty vector (A) or wild-type Bcl-2 (B) with or without azide treatment. C: percentage of cells with indicated plasmid transfection that had active Bax staining during azide-induced ATP depletion. Data are presented as means ± SD (n = 4). *Statistically significant difference from the treated group transfected with empty vector.

 
Conclusions Bcl-2 family proteins are considered the "master regulators" of apoptosis (7, 8, 14, 18, 19, 30). A key action site for these proteins within a cell is the mitochondria. Some Bcl-2 proteins such as Bak are localized in mitochondria, whereas others, for example Bax and Bid, normally are cytosolic but can move to mitochondria upon apoptotic stimulation. Yet, Bcl-2 itself has a dual subcellular localization, in both mitochondria and ER. Functionally, Bcl-2 in mitochondria is expected to sequester and neutralize the proapoptotic molecules. However, whether Bcl-2 localized in ER plays a role in the regulation of apoptosis is less clear. In this aspect, recent studies have shown that Bcl-2 may lower the Ca2+ store in ER and, as a result, reduce Ca2+ release from ER upon stimulation to inhibit apoptosis (21, 26). Nevertheless, it remains unclear whether this mechanism is universally applicable to other apoptotic models and whether ER Bcl-2 is directly involved. Earlier studies by Annis et al. (1) and Zhu and colleagues (36) suggested that the antiapoptotic activity of mitochondrial or ER-targeted Bcl-2 may depend on the cell type and experimental models examined. Thomenius et al. (29) further showed that Bcl-2 localized on ER could block Bax activation in mitochondria by sequestering Bad. In the current study, we addressed some of the related questions using renal proximal tubular cells. Our experiments show that mitochondrial Bcl-2 is much more effective than ER-targeted Bcl-2 in blocking Bax activation, cytochrome c release, and apoptosis following cisplatin treatment or ATP depletion of renal tubular cells. On the other hand, ER-targeted Bcl-2 is more effective in blocking ER stress-associated apoptosis. It is suggested that, depending on the apoptotic models, the subcellular localization of Bcl-2 is a key determinant of its antiapoptotic activity.


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This study was supported in part by grants from the National Institutes of Health and the Department of Veterans Affairs.


   ACKNOWLEDGMENTS
 
We thank Dr. David W. Andrews at McMaster University (Hamilton, Ontario, Canada) for the generous gift of the Bcl-2 constructs. We also thank Nikhil Prakash for assistance in some experiments during the graduate rotation period in our laboratory.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Z. Dong, Dept. of Cellular Biology and Anatomy, Medical College of Georgia and VA Medical Center, 1459 Laney Walker Blvd., Augusta, GA 30912 (e-mail: zdong{at}mail.mcg.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* K. Bhatt and L. Feng contributed equally to this study. Back


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