Potential conflict of interest: Nothing to report.
This work was supported by grants from the Howard Hughes Medical Institute (to Maria de Fátima Leite), Conselho Nacional de Desenvolvimento Científico e Tecnológico (to Maria de Fátima Leite, Rodrigo R. Resende, Gustavo B. Menezes, Valbert N. Cardoso, and Ana M. de Paula), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (to Maria de Fátima Leite, Rodrigo R. Resende, Gustavo B. Menezes, Valbert N. Cardoso, and Ana M. de Paula), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (to Mateus T. Guerra, Viviane A. Andrade, Marisa F. Casteluber, and Rodrigo R. Resende), Instituto Nacional de Ciência e Tecnologia (to Carla J. Aguiar), and the National Institutes of Health (DK57751, DK45710, and DK34989 to Michael H. Nathanson).
Subcellular Ca2+ signals control a variety of responses in the liver. For example, mitochondrial Ca2+ (Ca) regulates apoptosis, whereas Ca2+ in the nucleus regulates cell proliferation. Because apoptosis and cell growth can be related, we investigated whether Ca also affects liver regeneration. The Ca2+-buffering protein parvalbumin, which was targeted to the mitochondrial matrix and fused to green fluorescent protein, was expressed in the SKHep1 liver cell line; the vector was called parvalbumin–mitochondrial targeting sequence–green fluorescent protein (PV-MITO-GFP). This construct properly localized to and effectively buffered Ca2+ signals in the mitochondrial matrix. Additionally, the expression of PV-MITO-GFP reduced apoptosis induced by both intrinsic and extrinsic pathways. The reduction in cell death correlated with the increased expression of antiapoptotic genes [B cell lymphoma 2 (bcl-2), myeloid cell leukemia 1, and B cell lymphoma extra large] and with the decreased expression of proapoptotic genes [p53, B cell lymphoma 2–associated X protein (bax), apoptotic peptidase activating factor 1, and caspase-6]. PV-MITO-GFP was also expressed in hepatocytes in vivo with an adenoviral delivery system. Ca buffering in hepatocytes accelerated liver regeneration after partial hepatectomy, and this effect was associated with the increased expression of bcl-2 and the decreased expression of bax. Conclusion: Together, these results reveal an essential role for Ca in hepatocyte proliferation and liver regeneration, which may be mediated by the regulation of apoptosis. (HEPATOLOGY 2011;)
Liver regeneration is a complex process triggered by acute damage to the organ and can be induced experimentally by chemical or surgical injuries that result in a loss of parenchymal cells (i.e., hepatocytes).1 After partial hepatectomy (PH), liver mass restoration is achieved by a massive proliferation of hepatocytes, which switch from a quiescent phenotype to a proliferative phenotype. This cell growth response is driven by a number of cytokines and growth factors, such as interleukin-6,2 tumor necrosis factor (TNF),3 hepatocyte growth factor,4 and epidermal growth factor. Ca2+ signaling is one of the pathways activated during liver regeneration, and growth factors and hormones that promote Ca2+ release in hepatocytes, such as hepatocyte growth factor, epidermal growth factor, and vasopressin, are potent mitogens for this cell type.5-7
Ca2+ signaling regulates a variety of cellular functions in the liver; these functions range from bile secretion to cell proliferation.8, 9 This ability to regulate various functions is closely related to the subcellular compartments in which Ca2+ is released.10 For example, pericanalicular increases in Ca2+ regulate the targeting and canalicular insertion of multidrug resistance–associated protein 2,8 whereas nuclear Ca2+ signals regulate proliferation in liver cell lines.9 Mitochondria also participate in Ca2+ signaling. Mitochondrial Ca2+ (Ca) signals depend on cytosolic Ca2+ because there is a close association between inositol 1,4,5-trisphosphate receptors within the endoplasmic reticulum (ER) and mitochondria11; this permits the transmission of Ca2+ from the ER to the mitochondrial matrix.12 Ca signals regulate apoptosis in various cell systems.13, 14 This form of cell death is controlled in part by members of the B cell lymphoma 2 (Bcl-2) protein family, which directly modulate Ca2+ signaling.15 Proapoptotic members of this family induce cell death through either the enhancement of Ca2+ release from the ER or the facilitation of Ca2+ entry into mitochondria, which ultimately causes cytochrome C release and caspase activation. Conversely, prosurvival Bcl-2 proteins such as bcl-2, B cell lymphoma extra large (bcl-xL), and myeloid cell leukemia 1 (mcl-1) work either by the direct modulation of the activity of the inositol 1,4,5-trisphosphate receptor or by the reduction of Ca2+ entry into mitochondria, which prevents the generation of proapoptotic Ca2+ signals.16-18 However, this interplay between Ca and apoptosis has not been studied in the liver in the context of liver regeneration. Therefore, we investigated the role of Ca in the regulation of liver regeneration.
SKHep1 and HEK-293 cell lines were obtained from the American Type Culture Collection (Manassas, VA). Cells were grown at 37°C with 5% carbon dioxide/95% air in Dulbecco's modified Eagle's medium supplemented with 1% penicillin-streptomycin and 10% heat-inactivated fetal bovine serum (all from Gibco, Grand Island, NY). The pAc1GFP1-Mito vector, which directs the expression of a GFP-tagged protein to the mitochondrial matrix, was acquired from Clontech (Mountain View, CA). MitoTracker Red, Rhod-2/AM (fluorescent indicator of mitochondrial Ca2+), the SuperScript first-strand synthesis system for real-time polymerase chain reaction (PCR), PCR SuperMix, Lipofectamine, a caspase-9 detection kit, and antibodies against B cell lymphoma 2–associated X protein (bax), bcl-2, and c-Met were obtained from Invitrogen (Carlsbad, CA). Antibodies against β-actin, anti–γ-tubulin, adenosine triphosphate (ATP), and TNF-α were acquired from Sigma Aldrich (St. Louis, MO). Antibodies against proliferating cell nuclear antigen (PCNA) and epidermal growth factor receptor (EGFR) were obtained from Santa Cruz (Santa Cruz, CA) and Cell Signaling Technology (Boston, MA). Caspase-3 and caspase-8 detection kits were acquired from BD Biosciences (San Jose, CA). An apoptosis-inducing factor (AIF) reagent was obtained from Santa Cruz. Staurosporine (STA) was acquired from Calbiochem (San Diego, CA). All other reagents were of the highest quality that was commercially available.
Male Holtzman rats (40-50 g), which were obtained from CEBIO (Centro de Bioterismo, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil), were used for all studies. The animals were maintained on a standard diet and were housed with a 12-hour light-dark cycle. The investigation conformed to the standards of Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication 85-23, 1996 revision).
Plasmid and Adenovirus Constructs.
Complementary DNA (cDNA) for the Ca2+ binding protein parvalbumin (PV) was subcloned between the BamHI and AgeI restriction sites of the pAc1GFP1-Mito vector. The resulting vector encoded PV, which was fused to the mitochondrial targeting sequence (MTS) and green fluorescent protein (GFP), and it was called parvalbumin–mitochondrial targeting sequence–green fluorescent protein (PV-MITO-GFP). A recombinant adenovirus was used to deliver the parvalbumin–mitochondrial targeting sequence–green fluorescent protein construct (Ad-PV-Mito-GFP). The virus was amplified with HEK-293 cells and was purified with the VivaPure AdenoPack kit (Sartorius, Göttingen, Germany) according to the manufacturer's protocol. pAd-PV-MITO-GFP (3 × 109 pfu) was injected into rats by tail vein infusions, and the livers were processed at the indicated times.
Detection of Ca Signals.
Cells were perfused with ATP (1 μM), and Ca was monitored in SKHep1 cells with time-lapse confocal microscopy, as previously described.14 Transfected cells were identified with GFP fluorescence. MitoTracker Red and GFP colocalization images were collected as described previously.14
Protein lysates from SKHep1 cells or the total liver were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and were transferred to polyvinylidene fluoride membranes. Western blots were developed with the ECL Plus reagent. Densitometry was performed with ImageJ software (National Institutes of Health, Bethesda, MD).
For the determination of the proportion of dead cells, control SKHep1 cells and cells transfected with mitochondrial targeting sequence–green fluorescent protein (MITO-GFP) or PV-MITO-GFP were stimulated with 300 nM STA for 6 hours. The cells were trypsinized, fixed in 70% ethanol, and incubated with 0.5 mg/mL propidium iodide (PI). The cells were analyzed for GFP and PI fluorescence with the Becton Dickinson FACSCalibur system.
Total RNA was isolated from SKHep1 cells with TRIzol, and cDNA was synthesized with the SuperScript II kit (Invitrogen). DNA templates were amplified by real-time PCR with the StepOnePlus real-time PCR system (Applied Biosystems, Foster City, CA) and the SYBR Green method.19 β-Actin was used as an internal control to normalize variations in the cDNA content. Experiments were performed in triplicate for each data point. The sequences of the primers are listed in Supporting Table 1.
Apoptosis through the intrinsic pathway was induced by a 6-hour treatment with 300 nM STA. For the induction of apoptosis through the extrinsic pathway, cells were stimulated with 100 ng/mL TNF-α for 24 hours. Apoptosis was measured with caspase-3, caspase-8, and caspase-9 kits and colorimetric detection, as previously described.9, 19 Immunofluorescence for AIF was used to evaluate the caspase-independent intrinsic pathway in cells treated with 300 nM STA for 6 hours. Images were obtained with a Zeiss LSM 510 confocal microscope.
Measurement of Bromodeoxyuridine (BrdU) Incorporation.
Cell proliferation was measured by BrdU incorporation with an enzyme-linked immunosorbent assay (Roche Applied Science) according to the manufacturer's instructions. SKHep1 cells were plated onto 96-well culture plates, transfected with MITO-GFP or PV-MITO-GFP, and starved for 24 hours. The cells were then treated for 6 hours with 300 nM STA and were incubated 18 hours later with a BrdU labeling solution. BrdU incorporation was measured with a multiplate reader.
Intravital and Liver Section Confocal Microscopy.
Rat liver intravital microscopy was performed as described previously with modifications.20 Briefly, rats were anesthetized by an intraperitoneal injection of a mixture of 10 mg/kg xylazine hydrochloride and 200 mg/kg ketamine hydrochloride and were placed in a right lateral position on an adjustable microscope stage. A lateral abdominal incision was made to expose the liver surface, which was covered with a cover slip. The liver was visualized with an intravital multiphoton/confocal microscopy system based on a modified Olympus FV300 confocal microscope in an up-right configuration (a BX61 microscope). Images were obtained with the confocal laser at 488 nm or via multiphoton excitation at 840 nm with a UPlanFLN 10×/0.30 objective. For frozen liver section analysis, samples from rats injected with the adenovirus or saline were fixed, dehydrated in sucrose, and mounted for the visualization of GFP-positive cells with a Zeiss LSM 510 confocal microscope.
PH, Liver Histology, and Biochemical Analysis.
Two-thirds hepatectomy (i.e., PH) was performed on adult male Holtzman rats as described.21 One day before PH, the parvalbumin–mitochondrial targeting sequence–green fluorescent protein adenovirus (Ad-PV-MITO-GFP) was injected into the tail vein. For histology, 8-μm-thick liver cryostat sections were processed 24, 48, and 72 hours after PH for PCNA and hematoxylin-eosin staining. Serum samples were used to measure the levels of albumin, conjugated and total bilirubin, aminotransferases (aspartate aminotransferase and alanine aminotransferase), and alkaline phosphatase with commercial fluorometric kits according to the manufacturer's instructions.
Liver scintigraphy was performed with phytate labeled with technetium-99m (99mTc-phytate). Rats received 1.48 MBq of 99mTc-phytate via the tail vein. Fifteen minutes after the administration of the radiopharmaceutical, the animals were anesthetized and placed in the prone position on a gamma camera equipped with a low-energy collimator (Nuclide TH 22, Mediso, Budapest, Hungary). Ten-minute static planar images were acquired with a 256 pixel × 256 pixel matrix. The liver area (mm2) was determined by the amount of radioactivity uptake in the organ.
Determination of the Myeloperoxidase (MPO) Activity.
Neutrophil accumulation in the liver was quantified with MPO activity assays as previously described.22 MPO activity was assayed with measurements of the variation in the optical density (OD) at 450 nm with tetramethylbenzidine (1.6 mM) and hydrogen peroxide (0.5 mM). The results are expressed as relative neutrophil numbers, and they were calculated by comparisons of tissue supernatant OD values with the OD values of a standard neutrophil curve (>95% purity).
The results are expressed as means and standard errors of the mean. Prism (GraphPad Software, San Diego, CA) was used for data analysis. Statistical significance was tested with the Student t test or a one-way analysis of variance followed by Bonferroni posttests, and P < 0.05 was considered to indicate statistical significance.
Targeted PV as a Ca Buffer.
PV fused to an MTS and GFP was developed as a genetically encoded Ca buffer. GFP targeted to the mitochondrial matrix was used as a control (Fig. 1A). PV was effectively expressed in SKHep1 cells transfected with PV-MITO-GFP, as demonstrated by immunoblotting (Fig. 1B). Moreover, PV was correctly targeted to the mitochondrial matrix, as demonstrated by the colocalization of GFP and MitoTracker Red (Fig. 1C). For the evaluation of Ca2+ buffering by this construct, SKHep1 cells were stimulated with 1 μM ATP, and Ca was measured by Rhod-2/AM confocal microscopy. ATP elicited a robust increase in Ca in control cells and in cells expressing GFP alone, but this was reduced by approximately 90% in cells expressing PV in mitochondria (n = 3, P < 0.001; Fig. 2). These results demonstrated that PV-MITO-GFP was correctly targeted to the mitochondrial matrix and efficiently buffered agonist-induced Ca signals.
Ca Buffering Attenuates Apoptotic Cell Death.
Ca plays a crucial role in apoptosis, so we investigated the effect of Ca buffering on cell death. A treatment with STA increased the percentage of dead cells to 19.1% ± 3.7% (11.4% ± 0.7% for unstimulated cells, P < 0.001, n = 3). Upon Ca buffering, the rate of cell death induced by STA was reduced to 7.7% ± 2.2%, whereas the rate of cell death remained high (25.7% ± 1.8%) in cells transfected with MITO-GFP (P < 0.001, n = 3; Fig. 3A). The role of Ca in cell death was further characterized by the evaluation of the intrinsic or extrinsic apoptotic pathways because the two pathways converge at the level of Ca signaling.23 The intrinsic pathway was investigated through the measurement of caspase-9 and caspase-3 activity in SKHep1 cells stimulated with 100 nM STA for 6 hours. Caspase-9 activity was increased to 0.16% ± 0.06% after the STA treatment compared with 0.1% ± 0.02% in control cells, and this was blocked by the expression of PV-MITO-GFP (P < 0.001, n = 3; Fig. 3B). Similarly, STA-induced caspase-3 activity was inhibited by Ca buffering (Fig. 3C). Caspase-3 activity was increased from 43.0 ± 5.8 nmol/mg of protein in unstimulated cells to 97.5 ± 9.2 nmol/mg of protein in the STA-treated cells. STA increased caspase-3 activity in MITO-GFP cells to 126.2 ± 22.2 nmol/mg of protein, whereas the level of caspase-3 activity was 54.4 ± 6.4 nmol/mg of protein in SKHep1 cells expressing PV-MITO-GFP (P < 0.001, n = 3; Fig. 3C). Next, we investigated whether the caspase-independent intrinsic pathway was also affected by Ca buffering. Confocal immunofluorescence imaging of AIF demonstrated that targeting PV to mitochondria reduced the expression of this proapoptotic factor in comparison with SKHep1 cells transfected with the control construct MITO-GFP (Fig. 3D). These data show that the expression of PV in mitochondria protected cells from STA-induced cell death through the caspase-dependent and caspase-independent intrinsic apoptotic pathway. We also investigated whether PV-MITO affected the extrinsic apoptotic pathway. The activity of caspase-8 and caspase-3 was measured in control cells and in cells transfected with PV-MITO-GFP or MITO-GFP and treated with 100 ng/mL TNF-α for 6 hours. TNF-α increased caspase-8 and caspase-3 activity levels to 246.7 ± 15.2 and 63.3 ± 10.4 nmol/mg of protein, respectively; the levels of activity were 72.0 ± 2.6 and 25 ± 5 nmol/mg of protein, respectively, under control conditions. PV-MITO-GFP expression reduced the level of TNF-α–dependent caspase-8 activity to 150 ± 20 nmol/mg of protein (296.7 ± 30.5 nmol/mg of protein in MITO-GFP cells), and it completely abolished caspase-3 activity (P < 0.001, n = 3; Fig. 3E,F). These data demonstrate that Ca buffering also prevents apoptotic cell death through the extrinsic pathway.
Apoptosis can be modulated through the expression of antiapoptotic and proapoptotic genes,24 so we investigated whether alterations of Ca handling could affect the expression of such genes. Real-time PCR showed that Ca buffering reduced the expression of several proapoptotic genes under baseline or STA treatment conditions (Fig. 4A-D). The expression of each gene was normalized to its expression level in unstimulated, nontransfected cells. The expression of p53 was reduced to 0.72 ± 0.03 au in PV-MITO-GFP cells in comparison with the control (P < 0.001, n = 3). After the STA treatment, the expression of p53 increased to 2.2 ± 0.1 au in untransfected cells, whereas in PV-MITO-GFP cells, it remained at 1.08 ± 0.06 au (P < 0.001, n = 3; Fig. 4A). The expression of bax was reduced to 0.41 ± 0.04 au in PV-MITO-GFP cells in comparison with the control (P < 0.001), and after the STA treatment, the level of bax expression was 2.0 ± 0.2 au in nontransfected cells and 0.72 ± 0.06 au in PV-MITO-GFP cells (P < 0.001, n = 3; Fig. 4B). Although apoptotic peptidase activating factor 1 (apaf-1) expression was not altered between unstimulated control and transfected cells, after the STA treatment, apaf-1 expression increased to 1.69 ± 0.07 au in control cells and remained at 0.83 ± 0.10 au in PV-MITO-GFP cells (P < 0.001, n = 3; Fig. 4C). The baseline level of caspase-6 expression was reduced to 0.75 ± 0.036 au in PV-MITO-GFP cells in comparison with the control (P < 0.001), and it increased to 1.98 ± 0.09 au in nontransfected cells (0.97 ± 0.03 au in PV-MITO-GFP cells, P < 0.001, n = 3; Fig. 4D). Conversely, the expression of genes encoding antiapoptotic proteins was up-regulated after Ca buffering (Fig. 4E-G). Bcl-2 gene expression increased to 1.21 ± 0.13 au in PV-MITO-GFP cells in comparison with the control (P < 0.001, n = 3) and remained higher upon STA treatment (1.19 ± 0.17 versus 0.63 ± 0.09 au in the control, P < 0.001, n = 3; Fig. 4E). Similarly, the expression of mcl-1 and bcl-xL genes increased to 1.2 ± 0.06 and 1.41 ± 0.10 au, respectively, in PV-MITO-GFP cells and to 1.0 ± 0.05 and 1.0 ± 0.06 au, respectively, in the control (P < 0.001, n = 3). After the STA treatment, the expression levels of mcl-1 and bcl-xL remained high (1.18 ± 0.06 and 1.26 ± 0.10 au, respectively) in PV-MITO-GFP cells (0.46 ± 0.02 and 0.73 ± 0.06 au in the control, P < 0.001, n = 3; Fig. 4F,G). To examine whether the expression of these genes was also altered at the protein level in SKHep1 cells expressing PV-MITO-GFP, we performed immunoblotting for the antiapoptotic protein bcl-2 and the proapoptotic protein bax (Fig. 4H-J). The expression of the bcl-2 protein increased to 1.15 ± 0.09 au in cells expressing PV-MITO-GFP compared with 0.56 ± 0.08 au in control cells (P < 0.001, n = 3), whereas the expression of the bax protein decreased to 0.84 ± 0.09 au in cells expressing PV-MITO-GFP compared with 1.14 ± 0.09 au in control cells (P < 0.05, n = 3). Similar results were observed in cells treated with STA. These findings suggest that Ca buffering directs the expression ratio of proapoptotic and antiapoptotic protein members toward a predominantly antiapoptotic pathway.
For the determination of whether the decrease in cell death observed in PV-MITO-GFP cells was associated with changes in proliferation, SKHep1 cells were synchronized in G0 by serum withdrawal, transfected with the target constructs, and assayed for BrdU incorporation. No increase in cell proliferation was observed in PV-MITO-GFP cells in comparison with control cells or cells expressing MITO-GFP (supporting Fig. 1). However, with agonist-induced cell death, BrdU uptake was lower in cells expressing MITO-GFP versus cells expressing PV in the mitochondria (51.1% ± 5.3% for MITO-GFP versus 79.4% ± 3.6% for PV-MITO-GFP, P < 0.001, n = 3). Together, these results suggest that Ca buffering preferentially prevents cells from undergoing apoptosis instead of stimulating proliferation.
Ca Buffering Accelerates Liver Mass Restoration After PH.
Liver regeneration requires both increased cell proliferation and reduced apoptosis.25 The role of Ca signaling in apoptosis is well known, but its role in liver regeneration has not been studied. Therefore, we investigated the involvement of Ca in liver growth after two-thirds hepatectomy (i.e., PH). Adenoviruses encoding PV-MITO-GFP were injected into rats, and GFP fluorescence was used to monitor PV expression in the liver (Fig. 5A). PV was expressed throughout the liver lobule (Supporting Fig. 2). No fluorescence was observed in the livers of animals injected with a saline solution. Additionally, no GFP fluorescence was observed in the intestines of animals injected with Ad-PV-MITO-GFP (Supporting Fig. 3), and this demonstrated the preferential targeting of the vector to the liver. Confocal images of liver slices showed that PV was highly expressed in the mitochondria of hepatocytes (Fig. 5B). Moreover, immunoblotting confirmed PV expression in the liver after the adenovirus injection, whereas PV was not expressed in a control liver (Fig. 5C). Together, these data show that the injected adenoviruses efficiently delivered the Ca2+-buffering construct to hepatocytes in vivo and promoted the expression of PV in this cell type.
To investigate the role of Ca in liver regeneration, we performed PH 1 day after the Ad-PV-MITO-GFP injection, and liver regeneration was analyzed for 1 to 4 days after PH. The expression of PV-MITO-GFP significantly increased the liver area in comparison with the controls, mainly 24 to 48 hours after PH (Fig. 6). There was no difference in the liver area from day 1 to day 4 in sham-operated animals. However, 1 day after PH, the liver area was significantly smaller in PH rats versus animals injected with the adenovirus (198.1 ± 1.06 mm2 in the PH animals versus 257.2 ± 3.4 mm2 in the PH/Ad-PV-MITO-GFP animals, P < 0.05, n = 3). The difference in the liver area was even more pronounced 2 days after PH (208.2 ± 6.2 mm2 in the PH animals versus 340.1 ± 1.8 mm2 in the PH/Ad-PV-MITO-GFP animals, P < 0.001, n = 3). The PCNA labeling of liver slices similarly demonstrated that hepatocyte proliferation was accelerated by the buffering of Ca. The PCNA index peaked on day 1 in the PH/parvalbumin–mitochondrial targeting sequence adenovirus (Ad-PV-MITO) animals (61.2% ± 2.2%, n = 3); the PH animals showed the maximum PCNA index on day 2 (63.6% ± 4.0%, n = 3). These results indicate that Ca buffering accelerates liver regeneration after PH. This finding was further validated through measurements of the liver/body mass index after PH; this also demonstrated that liver regeneration occurred with accelerated kinetics in animals expressing PV in the mitochondria (Fig. 7A). One day after PH, the liver/body weight index was significantly smaller in PH rats versus PH animals injected with the adenovirus (54.8% ± 4.3% in the PH animals versus 63.2% ± 12.6% in the PH/Ad-PV-MITO-GFP animals, P < 0.01, n = 6). Two days after PH, the liver/body weight index was 72.3% ± 6.9% in the PH animals and 89.4% ± 8.8% in the PH/Ad-PV-MITO-GFP animals (P < 0.01, n = 6). The accelerated regeneration observed in the adenovirus-treated animals was not due to increased inflammation, as demonstrated by the measurement of the MPO activity and the histological examination of the livers from PH and PH/Ad-PV-MITO-GFP animals (Supporting Fig. 4). Liver weights returned to preoperative levels 3 days after PH. The liver histology and chemistry findings were similar in the control and PV-expressing animals 7 days after PH (Supporting Figs. 5 and 6). These data indicate that Ca homeostasis is important during liver regeneration.
To examine the mechanism by which Ca buffering accelerates liver regeneration, we investigated whether the expression of the antiapoptotic protein bcl-2 and the expression of the proapoptotic protein bax were altered in the livers of adenovirus-injected rats, as observed in SKHep1 cells. Ca buffering increased the expression of bcl-2 and reduced the expression of bax (Fig. 7B). Because growth factor signaling might affect the expression of apoptotic proteins during liver regeneration,4 the expression of receptors for two essential liver mitogens, EGFR and c-Met, was assessed. They showed similar levels in PH and PH/Ad-PV-MITO-GFP animals during the 3 days after PH (Fig. 7C-E). Together, these results suggest that Ca buffering promotes liver regeneration by inhibiting apoptosis.
Liver regeneration involves multiple factors and pathways1 that result in increased proliferation and decreased apoptosis of hepatocytes.25 Among the regulatory signaling pathways, a number of Ca2+-mobilizing agonists are known to contribute to liver regeneration.6, 26, 27 Hepatocytes respond to such agonists by altering intracellular Ca2+ signaling, which propagates throughout the liver as intercellular Ca2+ waves28, 29 that regulate several processes, including liver regeneration.6 Alterations in the Ca2+ signaling machinery have been reported to occur during liver regeneration,30 and although recent studies have examined the role of nuclear and cytosolic Ca2+ signals in cell proliferation,9, 31, 32 the impact of Ca on liver regeneration has not been directly investigated. Here we have examined the role of Ca during liver regeneration after PH, and we have found that Ca buffering, at least in part by inhibiting apoptosis, accelerates regeneration.
Mitochondria play an integral role in Ca2+ signaling and have a key function in most forms of apoptosis.33 Our results demonstrate that Ca buffering inhibits the intrinsic and extrinsic apoptotic pathways as well as the mitochondrial amplification loop; this was observed by the inhibition of caspase-8, caspase-9, and caspase-3 activation. This amplification is dependent on the release of cytochrome c from the mitochondrial matrix to the cytosol; there, it can further activate the effector caspases. Although we have not assessed the release of cytochrome C, we have found that Ca buffering inhibits the activation of caspase-9 by STA; this phenomenon is dependent on cytochrome C release from mitochondria.34 We have also found that Ca buffering inhibits caspase-independent but AIF-dependent cell death. This is consistent with previous observations showing that the dysregulation of Ca2+ homeostasis is a prerequisite for AIF-mediated apoptosis.35
Bcl-2 was the first gene identified as a regulator of apoptosis,36 and subsequently, several bcl-2 homologues were discovered that act as either proapoptotic or antiapoptotic effectors. The present data are in agreement with previous observations demonstrating that the overexpression of bcl-2, mcl-1, and bcl-xL37, 38 prevents cells from undergoing apoptosis, whereas bax, apaf-1, caspase-6, and p53 function to promote cell death.39 Ca buffering also shifted the Bax/Bcl-2 ratio toward the antiapoptotic profile, and this resulted in the accelerated restoration of liver mass after PH. This agrees with recent proteomic data showing that apoptosis pathways are inhibited during liver regeneration.40 Additionally, hepatocyte growth factor, an essential stimulus for liver regeneration, is known to have antiapoptotic activity in injured tissue.41 Similarly, TNF, another initiator of liver regeneration, also modulates apoptosis in addition to stimulating hepatocyte proliferation.42 Although our results suggest that Ca buffering accelerates liver regeneration by inhibiting apoptosis, an effect on cell proliferation cannot be entirely excluded because Bax/Bcl-2 family proteins regulate liver regeneration independently of their role in modulating apoptosis in the liver.43, 44 Moreover, Ca buffering might also accelerate liver regeneration by modulating ATP production in the mitochondrial matrix because the activity of enzymes of the tricarboxylic acid cycle is regulated by Ca2+.13
Heterologous expression of the Ca2+ binding protein PV has been widely used to study the role of Ca2+ signaling in the regulation of the cell cycle. PV was targeted to the nucleus or cytoplasm, and with this approach, the role of nuclear Ca2+ in regulating the cell cycle was established in a liver cell line.9 More recently, PV expression in the cytosol of hepatocytes in vivo demonstrated that cytosolic Ca2+ affects progression through the cell cycle after PH.32 Using PV targeted to the mitochondria, we have now shown that Ca also regulates liver regeneration. Future advances in this field should lead to a better understanding of the ways in which these various Ca2+ compartments act in an integrated manner to regulate liver regeneration.
The authors thank Gilson Nogueira for his technical support and Soraya Smaili for antibodies against Bax and Bcl-2 and useful discussions. The authors also thank Dawidson A. Gomes for assistance in the design of the parvalbumin construct. Confocal imaging was supported by CEMEL (Centro de Microscopia Eletrônica, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil).