The anti-apoptotic protein Mcl-1 inhibits mitochondrial Ca2+ signals.

Apoptosis contributes to the regulation of cell growth and regeneration and to the development of neoplasia. Mcl-1 is an anti-apoptotic protein that is particularly important for the development of hematological and biliary malignancies, but the mechanism of action of Mcl-1 is unknown. A number of pro- and anti-apoptotic proteins exhibit their effects by modulating Ca2+ signals, so we examined the effects of Mcl-1 on components of the Ca2+ signaling pathway that are known to regulate apoptosis. Expression of Mcl-1 did not affect expression of the inositol 1,4,5-trisphosphate receptor or the size of endoplasmic reticulum Ca2+ stores. However, mitochondrial Ca2+ signals induced by either Ca2+ agonists or apoptotic stimuli were decreased in cells overexpressing Mcl-1 and increased in cells in which Mcl-1 expression was inhibited. These findings provide evidence that Mcl-1 directly inhibits Ca2+ signals within mitochondria, which may provide a novel mechanism to inhibit apoptosis and thereby promote neoplasia.

Apoptosis can result from a range of extracellular or intracellular stimuli, which converge with activation of caspases to lead directly to cell death (1). Apoptosis provides a key control mechanism for the regulation of cell proliferation, differentiation and regeneration, and resistance to apoptosis has been linked to the development of neoplasia (2). Two general pathways to apoptosis have been described. One pathway is the so-called extrinsic pathway initiated by death receptors of the tumor necrosis factor receptor superfamily (3). The second pathway is the intrinsic pathway, which involves the convergence of intracellular stress signals on mitochondria resulting in mitochondrial permeability (4). In many cells, the death receptor pathway may also trigger mitochondrial dysfunction via caspase-mediated cleavage of the pro-apoptotic molecule, Bid, which when cleaved translocates to mitochondria triggering their permeabilization (5). In this respect, mitochondrial permeabilization is key to both pathways. Mitochondrial permeabilization results in release of cytochrome c into the cytosol, which then exhibits specific toxic effects (6,7). Apoptosis via the mitochondrial pathway is inhibited by members of the Bcl-2 family of proteins, which includes Bcl-2, Bcl-x L , and Mcl-1 (8 -10). Bcl-2 inhibits apoptosis in part by decreasing the size of Ca 2ϩ stores in the endoplasmic retic-ulum (ER) 2 (11), whereas Bcl-x L acts in part by inhibiting expression of the inositol 1,4,5-trisphosphate (InsP 3 ) receptor (InsP 3 R) (12), which is the principal ER Ca 2ϩ release channel in most types of cells.
Mcl-1 (myeloid cell leukemia-1) was first identified because this gene is up-regulated early in the differentiation of the ML-1 human myeloid leukemia cell line and was found to be a member of the emerging Bcl-2 gene family as well (13). However, the mechanism by which Mcl-1 inhibits apoptosis is not entirely understood. Because Mcl-1 is a member of the Bcl-2 family, we examined the effects of this protein on Ca 2ϩ signaling pathways.

EXPERIMENTAL PROCEDURES
Materials, Reagents, and Cell Lines-The Ca 2ϩ dyes fluo-4, magfluo-4, and rhod-2, the mitochondrial dye MitoTracker green, and the nuclear stain TO-PRO-3 were from Molecular Probes (Eugene, OR). JC-1, ATP, and staurosporine were from Sigma. A monoclonal antibody labeling the N-terminal region of the human type III InsP 3 R was from BD Transduction Laboratories (Lexington, KY) (14). A polyclonal antibody for Mcl-1 was from Santa Cruz Biotechnology (Santa Cruz, CA). A monoclonal antibody for the 17-kDa subunit of oxidative phosphorylation complex I, which is localized to mitochondria, was from Molecular Probes. The secondary antibodies were Alexa 488 anti-rabbit, Alexa 568 anti-mouse, and Alexa 647 anti-rabbit IgG (Molecular Probes). Mz-Cha-1 cells derived from a human biliary adenocarcinoma (15) were kindly provided by Dr. Greg Fitz (University of Texas Southwestern). Cells were maintained at 37°C with 5% CO 2 in minimal essential medium-␣ supplemented with 10% fetal bovine serum, 1% penicillin, gentamycin and streptomycin, and 2 mM glutamine. HEK 293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum plus antibiotics.
Immunofluorescence-Immunochemistry was performed on Mz-Cha-1 cells and paraffin-embedded sections of human liver biopsy specimens as described previously (16). Briefly, Mz-Cha-1 Cells were fixed in 3.7% formaldehyde and then permeabilized in 0.1% Triton X-100. Paraffin-embedded sections were pretreated with 1 mM citrate buffer at 100°C. Primary antibodies used were anti-type III IP3R-3 mouse monoclonal (1:100) and anti-Mcl-1 rabbit polyclonal (1:100). Secondary antibodies were Alexa 488 anti-rabbit (1:500), Alexa 568 anti-mouse (1:500), and Alexa 647 anti-rabbit (1:500). Cells also were labeled with the nuclear stain TO-PRO-3. Negative controls were performed under each experimental condition by incubating tissue with secondary antibodies but not primary antibodies. All images were collected using a Zeiss LSM 510 Laser Scanning Confocal Microscope (Thornwood, NY). To ensure specificity of staining, images were obtained using machine settings at which no fluorescence was detectable in negative control samples. Immunofluorescence images were obtained by excitation at 488 nm with observation at 505-550 nm to detect Alexa 488 or GFP, by excitation at 543 nm and observed at Ͼ585 nm to detect Alexa 568 or DsRed, then by excitation at 633 nm with observation at Ͼ650 nm to detect Alexa 647 or TO-PRO-3. Mcl-1 or InsP 3 R immunofluorescence was quantified in transfected Mz-Cha-1 cells by normalizing the fluorescence relative to the fluorescence detected in nearby non-transfected cells. To perform this calculation, transfected cells in each microscopic field were identified by expression of DsRed or GFP. Mean fluorescence in each transfected cell then was divided by mean fluorescence measured in at least five non-transfected cells in the same microscopic field, and the ratio was multiplied by 100%.
Plasmids and Transfections-The plasmids for Mcl-1 cDNA and siRNA have been described previously (17). In particular, a specific double-stranded 21-nucleotide RNA sequence homologous to the target message was used to silence Mcl-1 (17). Cells were co-transfected with enhanced green fluorescent protein (GFP) or DsRed (both from Clontech) to identify transfected cells. Transfections were carried out using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instruction. Transfection efficiency was typically 5-10%. Cells were used 48 h after transfection.
Mitochondrial Preparation and Cytosolic Extracts-Mitochondria were isolated using a commercially available mitochondria isolation kit (Sigma) according to the manufacturer's instructions. Briefly, mitochondria were prepared from cells by homogenization followed by low speed (600 ϫ g) and then high speed (1100 ϫ g) centrifugation. The final pellet contained the crude mitochondrial fraction that was used for immunoblots. Purity of the mitochondrial fraction was validated by a fluorometric assay that measures uptake of the cationic carbocyanine dye JC-1 by intact mitochondria (18). In selected experiments, JC-1 was used to measure mitochondrial membrane potential directly in intact Mz-Cha-1. Cells were incubated for 30 min at 37°C with JC-1 (10 g/ml), and then examined by confocal microscopy. Fluorescence images were obtained by excitation at 488 nm with observation at 505-550 nm and at Ͼ585 nm to detect potential-sensitive color shifts.
Calcium Measurements-Free mitochondrial Ca 2ϩ (Ca m 2ϩ ) was measured in wild type Mz-Cha-1 cells and in Mz-Cha-1 cells transiently transfected with Mcl-1 cDNA or Mcl-1-specific siRNA using the mitochondrial Ca 2ϩ dye rhod-2 and time lapse confocal microscopy. rhod-2 can sometimes detect cytosolic rather than mitochondrial Ca 2ϩ , but rhod-2 labeling in Mz-Cha-1 cells was punctuate, and Ca 2ϩ signals detected by rhod-2 were different from those detected by the cytosolic Ca 2ϩ dye fluo-4 (not shown), suggesting that rhod-2 fluorescence reflected Ca m 2ϩ in these cells. Cells were incubated for 30 min at 37°C with rhod-2/AM (10 M). Coverslips containing the cells were transferred to a custom-built perfusion chamber on the stage of a Bio-Rad MRC-1024 confocal microscope (Hercules, CA) (14,16,19). Ca m 2ϩ was monitored in these cells by exciting the specimens at 543 nm and observing at Ͼ585 nm to detect rhod-2 emission signals. Cells were stimulated with ATP (10 or 100 M) or staurosporine (1 M), and images were acquired every 150 -600 ms. Increases in Ca m 2ϩ were expressed as percent increases in fluorescence intensity of rhod-2. In separate studies, ER Ca 2ϩ was measured using the low affinity Ca 2ϩ dye mag-fluo-4 (K d 22 M) and examined by confocal microscopy (22). Cells were incubated for 30 min at 37°C with mag-fluo-4/AM (6 M) then observed using the same confocal imaging system used to detect rhod-2. ER Ca 2ϩ was monitored in these cells by exciting the specimens at 488 nm and observing at Ͼ515 nm to detect mag-fluo-4 emission signals. Cells were stimulated with thapsigargin (2 M) to deplete ER Ca 2ϩ stores, and images were acquired every 1-2 s. Changes in ER Ca 2ϩ were expressed as percent increases or decreases in fluorescence intensity of mag-fluo-4. rhod-2 and mag-fluo-4 fluorescence was quantified in transfected Mz-Cha-1 cells by normalizing the fluorescence relative to Confocal immunofluorescence images were obtained from paraffinembedded liver biopsy or surgical resection specimens. Each tissue section was double-labeled with antibodies against Mcl-1 (green) and type III InsP 3 R (red). A, normal liver. Note that the Mcl-1 is distributed diffusely throughout the cytosol, while the InsP 3 R is localized to the apical region. No colocalization is seen. B, cholangiocarcinoma. The subcellular distribution of both Mcl-1 and the InsP 3 R is similar to what is observed in normal cholangiocytes, although the staining for each is more intense. C, bile duct obstruction. Neither Mcl-1 nor the InsP 3 R is detectable. Scale bar, 10 m. the fluorescence detected in nearby non-transfected cells. To perform this calculation, transfected cells in each microscopic field were identified by expression of GFP (in cells loaded with rhod-2) or DsRed (in cells loaded with mag-fluo-4). Mean fluorescence in each transfected cell then was divided by mean fluorescence measured in at least five nontransfected cells in the same microscopic field, and the ratio was multiplied by 100%.
Statistical Analysis-Data are represented as mean Ϯ S.E. Means between groups were compared using Student's t test. p Ͻ 0.05 was taken to indicate statistically significant differences.

Expression of Mcl-1 and Type III InsP 3 R in Primary Cholangiocytes and in a Cholangiocarcinoma
Cell Line-Mcl-1 is thought to play a role in development of cholangiocarcinoma (17), but expression of this protein has not been investigated in primary biliary tissue. Therefore we used confocal immunofluorescence to examine expression of Mcl-1 in liver biopsies from eight patients with normal bile ducts, five patients with biliary obstruction due to stone disease (n ϭ 4) or malignancy (n ϭ 1), and five patients with cholangiocarcinoma ( Fig. 1). Specimens were co-labeled for the type III InsP 3 R (InsP 3 R-3). This isoform accounts for Ͼ80% of InsP 3 Rs in bile ducts and is known to be concentrated in the apical region (16,20). Mcl-1 labeling was present in normal cholangio-cytes and was distributed diffusely throughout the cytosol, whereas InsP 3 R-3 was concentrated apically as shown previously (Fig. 1A). Both Mcl-1 and InsP 3 R-3 were detected in cholangiocarcinoma as well (Fig.  1B). Quantitative immunofluorescence suggested that Mcl-1 expression was increased in cholangiocarcinoma, because fluorescence increased from 86 Ϯ 1 pixel values in normal cholangiocytes to 124 Ϯ 2    pixel values (p Ͻ 0.0005, mean Ϯ S.E. of fluorescence intensity obtained using the same confocal machine settings for each sample). Mcl-1 expression was decreased to 58 Ϯ 1 pixel values, and InsP 3 R-3 expression was nearly absent in patients with biliary obstruction (p Ͻ 0.0001 relative to normal tissue, Fig. 1C). Interpretation of these results is limited, because there was insufficient tissue from these patient specimens to perform quantitative immunoblots. However, these results demonstrate that Mcl-1 is expressed in primary human cholangiocytes and suggest that expression is increased in cholangiocarcinoma. Next, we examined the expression and distribution of Mcl-1 and InsP 3 R-3 in Mz-ChA-1 cells, because this is a well characterized human cholangiocarcinoma cell line (21) and InsP 3 R-3 accounts for nearly all of the InsP 3 R in these cells (data not shown). Mcl-1 was distributed throughout the cytosol, whereas InsP 3 R-3 was distributed in a perinuclear pattern. The two proteins did not co-localize (Fig. 2). Based on these findings, Mz-Cha-1 cells were used as the model cell line for the remaining studies.
Mcl-1 Does Not Alter Expression of the InsP 3 Receptor-Bcl-x L has been shown to inhibit apoptosis in part by decreasing expression of the InsP 3 R (12), so we examined whether Mcl-1 similarly alters InsP 3 R   (Fig. 3). HEK293 cells were used to validate the Mcl-1 cDNA and siRNA constructs (Fig. 3), and all subsequent transfections were performed in Mz-Cha-1 cells. Cells were co-transfected with DsRed as a marker of successful transfection. Control cells were transfected with DsRed alone. Expression of Mcl-1 and InsP 3 R in each group was evaluated simultaneously by quantitative confocal immunofluorescence (20) and was normalized by expression of these proteins in nontransfected cells on the same coverslips (Figs. 4 and 5). Mcl-1 fluorescence in the DsRed control group was 100 Ϯ 12% of the fluorescence in non-transfected cells in co-culture, whereas fluorescence staining of InsP 3 R-3 was 101 Ϯ 11%. The normalized Mcl-1 fluorescence in siRNAtreated cells was reduced to 54 Ϯ 4% of that of non-transfected cells in co-culture (p Ͻ 0.005 versus control), whereas that of InsP 3 R-3 in these cells was 92 Ϯ 6% (p Ͼ 0.1 versus control). The fluorescence intensity of Mcl-1 labeling in cells transfected to overexpress this protein was 193 Ϯ 21% of that seen in non-transfected cells in co-culture (p Ͻ 0.005 versus control), whereas that of InsP 3 R-3 was 102 Ϯ 12% (p Ͼ 0.1 versus control). Thus, neither increased nor decreased expression of Mcl-1 altered the expression of the InsP 3 R. To analyze these results further, we exam-  (Fig. 6D), providing further evidence of selective labeling of ER Ca 2ϩ stores. Treatment with thapsigargin induced a similar decrease in fluorescence in all (control, siRNA, and cDNA) transfected cells as well as in non-transfected cells (p Ͼ 0.8 by analysis of variance, Fig. 6E). These results provide evidence that, unlike Bcl-2, Mcl-1 does not affect the size of ER calcium stores.
Mcl-1 Inhibits Mitochondrial Calcium Signaling-Immunoblot analysis of mitochondrial and non-mitochondrial fractions of Mz-Cha-1 cells demonstrated that Mcl-1 is localized to the mitochondria (Fig. 7A), consistent with previous reports (23). Confocal immunofluorescence of Mcl-1 was performed to further confirm this observation (Fig. 7B).
Mcl-1 co-localized with oxidative phosphorylation complex I, which is expressed only in mitochondria. This provides additional evidence that Mcl-1 is mitochondrial, so we examined the effects of Mcl-1 on mitochondrial Ca 2ϩ signals. Excessive increases in free mitochondrial Ca 2ϩ can lead to formation of the permeability transition pore (25) and can also release cytochrome c and induce apoptosis (26). To determine whether Mcl-1 influences free mitochondrial Ca 2ϩ signals, Mz-ChA-1  Cells were loaded with the cationic Ca 2ϩ dye rhod-2 (red) and the mitochondrion-specific dye MitoTracker green (green). Confocal image demonstrates that the two dyes co-localize (yellow), indicating that rhod-2 selectively labels mitochondria in this cell type. cells were loaded with the mitochondrial Ca 2ϩ dye rhod-2 (27,28) and examined by confocal microscopy. To confirm the subcellular distribution of rhod-2, a subset of cells were loaded with both rhod-2 and the mitochondrial dye MitoTracker green (Fig. 8). Confocal imaging demonstrated that the two dyes co-localized, which demonstrates that rhod-2 preferentially labels mitochondria in this cell type. Cells were then transfected with either Mcl-1 cDNA or siRNA for Mcl-1, along with GFP as a marker of successful transfection. Control cells were transfected with GFP alone. As an additional control, mitochondrial Ca 2ϩ signals in all transfected cells were compared with signals observed in co-cultured non-transfected cells on the same coverslips. Cells were stimulated with ATP (10 and 100 M), which is known to increase Ca 2ϩ via P2Y receptor-mediated InsP 3 formation in this cell type (21,29). Mitochondrial Ca 2ϩ increased just as often in cells transfected with GFP alone as in matched non-transfected controls (p Ͼ 0.1, Fig. 9, B and C). In both groups, Ca 2ϩ increased in most cells stimulated with 100 M ATP (69 Ϯ 14% and 75 Ϯ 11%, respectively), but only in a minority of cells stimulated with 10 M ATP (18 Ϯ 11% and 26 Ϯ 11%). In contrast, mitochondrial Ca 2ϩ increased less often in cells overexpressing Mcl-1 than in non-transfected controls, regardless of the concentration of ATP. Specifically, mitochondrial Ca 2ϩ increased in a minority of overexpressing cells stimulated with 100 M ATP (14 Ϯ 9% versus 82 Ϯ 9%, p Ͻ 0.0005), and increased in a minimal percent of cells stimulated with 10 M ATP (1 Ϯ 1% versus 30 Ϯ 10%, p Ͻ 0.05). Mitochondrial Ca 2ϩ increased in most cells in which Mcl-1 expression was decreased, regardless of the ATP concentration. Thus, mitochondrial Ca 2ϩ increased more often than in non-transfected controls when cells were stimulated with 10 M ATP (64 Ϯ 12% versus 26 Ϯ 13%, p Ͻ 0.05) but not 100 M ATP (84 Ϯ 7% versus 73 Ϯ 11%, p Ͼ 0.1). These findings demonstrate that Mcl-1 inhibits mitochondrial Ca 2ϩ signaling.
Mitochondrial Ca 2ϩ also was examined during exposure to an apoptotic stimulus. Mz-ChA-1 cells were loaded with rhod-2 and examined by confocal microscopy as above. Here, though, cells were transfected with either Mcl-1 cDNA or siRNA for Mcl-1, along with a GFP-tagged cytochrome c to serve as a marker of successful transfection as well as an index of apoptosis. Control cells were transfected with GFP-cytochrome c alone. As an additional control, mitochondrial Ca 2ϩ signals in all transfected cells were compared with signals observed in co-cultured non-transfected cells on the same coverslips. Cells were stimulated with staurosporine (1 M) to induce apoptosis. Staurosporine increased mitochondrial Ca 2ϩ in 5 of 6 cells in which Mcl-1 expression was reduced (Fig. 10A), and in none of 5 cells in which Mcl-1 was overexpressed (Fig. 10B). For comparison, staurosporine increased mitochondrial Ca 2ϩ in 2 of 6 cells transfected with GFP-cytochrome c alone, which is similar to the frequency with which Ca 2ϩ signals were observed in non-transfected cells (26 of 109 cells, or 24%). Next, the subcellular localization of rhod-2 and GFP-cytochrome c was monitored in transfected cells before and 3 h after treatment with staurosporine. rhod-2 and GFP-cytochrome c were co-localized prior to treatment (not shown). The two labels no longer were co-localized after treatment in cells in which Mcl-1 expression was decreased (Fig. 10C), indicating the loss of cytochrome c from mitochondria that is associated with development of apoptosis. In contrast, co-localization persisted in cells in which Mcl-1 expression was increased (Fig. 10C), consistent with protection against apoptosis. Because of the specific mitochondrial actions of Mcl-1, we examined the effects of Mcl-1 expression on mitochondrial membrane potential using the cationic potential-sensitive dye JC-1.  fluorescence in non-transfected cells, p Ͼ 0.3), siRNA (98 Ϯ 7% of non-transfected cells, p Ͼ 0.4), or GFP alone (106 Ϯ 7%, p Ͼ 0.25). These findings demonstrate that Mcl-1 inhibits the mitochondrial Ca 2ϩ signaling that is associated with development of apoptosis and suggest that this effect is not due to changes in mitochondrial membrane potential.

DISCUSSION
Mitochondria play an integral role in Ca 2ϩ signaling pathways and patterns. Mitochondria are in close apposition to InsP 3 Rs (30) and thus are exposed to more abrupt and intense increases in Ca 2ϩ than occurs in most regions of the cytosol (30). Because mitochondria take up Ca 2ϩ via a potential-driven uniporter (31), this spatial arrangement enables mitochondria to modulate increases in cytosolic Ca 2ϩ . For example, oxidizable substrates that energize mitochondria increase the ampli-tude of Ca 2ϩ signals and the speed of Ca 2ϩ waves in the cytosol, whereas inhibitors of oxidation such as mitochondrial uncouplers dampen cytosolic Ca 2ϩ signals (32). The Ca 2ϩ signals that are transmitted into mitochondria have specific mitochondrial effects as well, such as regulation of NADPH formation (27). However, excessive increases in Ca m 2ϩ result in formation of the permeability transition pore (PTP), which can be either transient or prolonged (25). PTP formation permits rapid loss of Ca 2ϩ from the mitochondria to the cytosol, so that this pathway may enable Ca 2ϩ waves to spread across neighboring mitochondria in a fashion analogous to Ca 2ϩ -induced Ca 2ϩ release (25). PTP formation also results in loss of ATP, but resealing of the pore permits ATP production to be restored, along with preservation of associated cellular metabolic activity (34). In contrast, prolonged formation of the PTP permits progression to cell necrosis (35,36). Apoptosis may result from increases in mitochondrial permeability that occur independent of PTP formation, because mitochondria from cyclophilin D knock-out mice cannot form PTP and cells from these mice are resistant to necrosis but do not have altered susceptibility to apoptosis (35,36). Although the mechanism remains unclear, progression to apoptosis includes an increase in mitochondrial permeability that results in leakage of cytochrome c into the cytosol (34). Under these circumstances, "death waves" spread across neighboring mitochondria throughout the cell, leaving depolarized mitochondria and leakage of cytochrome c in their wake (24). The findings that Mcl-1 is localized to mitochondria and inhibits mitochondrial Ca 2ϩ signals suggest that this protein may inhibit apoptosis via direct inhibition of these death waves.
Ca 2ϩ is involved in several critical steps in the induction of apoptosis, including activation of caspases (4) plus PTP formation as noted above (37). Ca 2ϩ release from the InsP 3 R is a particularly important step for inducing apoptosis. Initial evidence suggested that the type III InsP 3 R is more effective than the type I isoform in inducing apoptosis (38), although subsequent work suggested the type I InsP 3 R can mediate apoptosis as well (39). The ability of the InsP 3 R to modulate apoptosis is related to its actions as a Ca 2ϩ release channel. Ca 2ϩ released from the InsP 3 R can be taken up by neighboring mitochondria and lead to cytochrome c release. Cytochrome c that has leaked from mitochondria then binds directly to the InsP 3 R, which blocks the Ca 2ϩ -induced inhibition of the receptor that occurs at high cytosolic Ca 2ϩ concentrations (26). This leads to a positive feedback loop whereby enhanced InsP 3 -medi-  ated Ca 2ϩ release causes further Ca 2ϩ overload of mitochondria, leading to further leakage of cytochrome c and then further enhancement of InsP 3 -mediated Ca 2ϩ release (26). Conversely, factors that inhibit InsP 3mediated Ca 2ϩ release lead to inhibition of apoptosis. For example, the mitochondrial stress pathway leading to apoptosis activates nuclear factor of activated T cells, which in turn binds to the InsP 3 R promoter and increases expression of the InsP 3 R. The anti-apoptotic protein Bcl-x L reduces binding of nuclear factor of activated T cells to DNA, resulting in decreased InsP 3 R expression (12). This in turn reduces InsP 3 -mediated Ca 2ϩ release to protect against apoptosis (12). In contrast to Bcl-x L , Bcl-2 instead increases leakage of Ca 2ϩ from the ER, but this also results in decreased Ca 2ϩ signals in both the cytosol and mitochondria (11). Recent evidence suggests that the pro-apoptotic protein Bax exhibits an effect opposite to that of Bcl-2; Bax increases Ca 2ϩ loading in the ER, which enhances transmission of Ca 2ϩ signals to the mitochondria (40). Mcl-1 is thought to be the principal Bcl-2 family member in cholangiocytes (17), but little is known about the mechanism by which it inhibits apoptosis. Both apoptosis (41) and InsP 3 R expression (16) are decreased in biliary obstruction, so one hypothesis would be that Mcl-1, like Bclx L , inhibits expression of the InsP 3 R. However, we found that neither InsP 3 R expression nor ER Ca 2ϩ stores are affected by Mcl-1. Rather, our findings show that Mcl-1 inhibits Ca 2ϩ signaling directly within mitochondria. This suggests that Bcl-2 family members inhibit apoptosis through a range of complementary effects on Ca 2ϩ signaling pathways (Fig. 11).
Mcl-1 inhibits cell death, and the survival of patients is decreased in leukemias and lymphomas in which Mcl-1 expression is increased (42,43). Mcl-1 expression may be important for development of cholangiocarcinoma as well (17), and the current work provides evidence that Mcl-1 expression is increased in this type of malignancy. Mcl-1 inhibits apoptosis in part by inhibition of the pro-apoptotic protein Bak. Mcl-1 binds to Bak, which renders Bak inactive by maintaining it as a monomer (6). The tumor suppressor p53 promotes apoptosis by dissociating Mcl-1 from Bak, while facilitating Bak to oligomerize into its activated form (6). Bak can localize to either the ER or mitochondria, and ER Bak indirectly affects Ca m 2ϩ by depleting ER Ca 2ϩ stores (28). However, mitochondrial Bak may protect against apoptosis in a fashion that is independent of Ca m 2ϩ (28). Further studies will be needed to understand whether Mcl-1 directly inhibits increases in Ca m 2ϩ or whether Mcl-1 indirectly inhibits Ca m 2ϩ by rendering Bak in an inactive conformation.