Calcium-Mediated Activation of c-Jun NH2-Terminal Kinase (JNK) and Apoptosis in Response to Cadmium in Murine Macrophages

Jiyoung Kim and Raghubir P. Sharma1

Interdisciplinary Program of Toxicology, Department of Physiology and Pharmacology, College of Veterinary Medicine, The University of Georgia, Athens, Georgia

Received May 11, 2004; accepted July 2, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cadmium is a well-known carcinogenic and immunotoxic metal commonly found in cigarette smoke and industrial effluent. An altered intracellular calcium ([Ca2+]i) level has been implicated in the pathophysiology of immune dysfunction. The present study was designed to determine the possible involvement of calcium (Ca2+) and mitogen-activated protein kinases (MAPKs) signaling pathways on cadmium-induced cell death in J774A.1 murine macrophage cells. Cadmium caused a low-amplitude [Ca2+]i elevation at 20 µM and rapid and high-amplitude [Ca2+]i elevation at 500 µM. Exposure to cadmium dose-dependently induced phosphorylation of c-Jun NH2-terminal kinase (JNK) and deactivated p38 MAPK. Use of the selective JNK inhibitor SP600125 suggested that activation of JNK is pro-apoptotic and pro-necrotic. Buffering of the calcium response with 1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxy-methyl) ester (BAPTA-AM) and ethylene glycol-bis-(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) completely blocked cadmium-induced apoptotic response. The pretreatment of cells with BAPTA-AM and EGTA suppressed the cadmium-induced cell injury, including growth arrest, mitochondrial activity impairment, and necrosis, and it also recovered the cadmium-altered JNK and p38 MAPK activity. Chelating [Ca2+]i also reversed cadmium-induced hydrogen peroxide generation, suggesting that production of reactive oxygen species (ROS) is related to [Ca2+]i. The present study showed that cadmium induces a [Ca2+]i-ROS-JNK-caspase-3 signaling pathway leading to apoptosis. Furthermore, cadmium-induced [Ca2+]i regulates phosphorylation/dephosphorylation of JNK and p38, and it modulates signal transduction pathways to proliferation, mitochondrial activity, and necrosis.

Key Words: cadmium; calcium; ROS; MAPKs; growth arrest; apoptosis.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cadmium is a naturally occurring nonessential and toxic heavy metal commonly found in stabilizers in polyvinyl chloride products, color pigment, several alloys and, most commonly, in re-chargeable nickel-cadmium batteries (Jarup, 2003Go). Cigarette smoking may cause significant increases in the blood cadmium level, and it has been reported that smokers have 4–5 times higher levels of cadmium in blood than non-smokers (Jarup et al., 1998Go). Inhalation of cadmium through tobacco smoking will directly affect the respiratory system without first-pass elimination, and an elevated blood level of cadmium may be a factor in immunodepression in smokers. Cadmium causes apoptotic cell death in NIH 3T3 murine fibroblasts (Biagioli et al., 2001Go), in CCRF-CEM human T-cell line (Iryo et al., 2000Go), and in rat testicular tissues (Xu et al., 1996Go). The International Agency for Research on Cancer (IARC) has classified cadmium as carcinogenic to humans (group 1), based on sufficient evidence for carcinogenicity in both human and animal studies (IARC, 1993Go).

Calcium ions are central to multiple signal transduction pathways to accomplish a variety of biological functions. The spatial and temporal regulation of intracellular calcium ([Ca2+]i) serves as a modulator of pathways involved in learning and memory, fertilization, proliferation, and development (Berridge et al., 2000Go). However, high [Ca2+]i can cause disruption of mitochondrial Ca2+ equilibrium, which results in reactive oxygen species (ROS) formation due to the stimulation of electron flux along the electron transport chain (Chacon and Acosta, 1991Go). Under oxidative stress, mitochondrial Ca2+ accumulation can switch from physiologically beneficial process to cell death signal (Ermak and Davies, 2001Go). Ca2+-dependent processes induced by cadmium (<1 µM) activate p21ras-dependent MAPK pathways, and nuclear factor-{kappa}B (NF-{kappa}B)–dependent gene expression, to stimulate proliferation in peritoneal macrophages (Misra et al., 2002Go). Besides the direct interaction of cadmium with intracellular molecules, altered [Ca2+]i homeostasis has been considered as a target of toxic action by cadmium.

Cd2+ and Ca2+ are two closely related elements with similarity in many aspects, partially because of their similar ionic radii; the radius of a common form of free Cd2+ in the body and that of Ca2+ are 0.099 and 0.097 nm, respectively (Weast and Astle, 1982Go). Cadmium is a potent Ca2+ channel blocker and inhibits Ca2+ cellular uptake (Thevenod and Jones, 1992Go). Cadmium has a high affinity for and activates calmodulin, a Ca2+-binding protein that regulates a variety enzymes and cell progresses (Behra and Gall., 1991Go). Moreover, a number of recent studies demonstrated that cadmium interacts with the function of Ca2+-dependent enzymes such as endonuclease and regulatory proteins such as protein kinase C (PKC) and phospholipase C, thus interfering with the Ca2+-signaling pathways (Lohmann and Beyersmann, 1993Go; Long, 1997Go; Misra et al., 2002Go).

Mitogen-activated protein kinases (MAPKs) belong to a family of Ser/Thr protein kinases that transmit extracellular signals into the nucleus. There are three subfamilies of MAPKs including c-Jun NH2-terminal kinase (JNK; also known as stress-activated protein kinase), p38 MAPK, and extracellular signal–related kinase (ERK) (Schaeffer and Weber, 1999Go). These MAPKs are believed to be important biomolecules in cell differentiation, cell movement, cell division, and cell death induced by extracellular stimuli (Schaeffer and Weber, 1999Go). We recently reported ERK signaling–dependent G2/M arrest and cell death in murine macrophages by cadmium (Kim et al., 2003Go). However, it is not clear if cadmium-altered MAPK activity is interrelated with Ca2+. Intracellular calcium elevation by cadmium was required for JNK activation in LLC-PK1 cells (Matsuoka and Igisu, 1998Go). Calcium-dependent PKC activation by serotonin contributed to ERK phosphorylation in the nudibranch mollusk Hermissenda (Crow et al., 2001Go). Elevated [Ca2+]i was involved in p38 MAPK-induced neuronal cell death by pneumolysin (Stringaris et al., 2002Go). These findings suggest that the role/activation of ERK, JNK, and p38 may be connected with [Ca2+]i.

Based on substantial evidence, mostly from in vivo animal models, cadmium is able to damage both the humoral immune response and cell-mediated immunity (Descotes, 1992Go). J774A.1 cells are a commonly used murine macrophage cell line which possesses similarities to mature macrophages, making them an alternative to primary cells (Yan and Cirillo, 2004Go). J774A.1 cells originated from BALB/c mouse reticulum cell sarcoma, and because of their tumor-like property, these cells may be resistant to cadmium toxicity. Studies using macrophages on cadmium-induced inhibition of growth progression and subsequent cell death are helpful to understand cadmium's effect on the immune system. There have been studies to show that elevated [Ca2+]i by cadmium mediates several different signaling pathways depending on cell types. However, the kinetics of [Ca2+]i elevation and exact involvement of [Ca2+]i on cadmium toxicity, including cell death, growth arrest, mitochondrial activity, and ROS generation has not been clearly determined in macrophages. We have proved the role of intracellular and extracellular Ca2+ in cadmium-altered JNK and p38 MAPK, and the importance of JNK activation on cadmium-induced activation of caspase-3 and apoptosis in murine macrophages.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Reagents. Cadmium (CdCl2, Sigma Chemical Co., St. Louis, MO) was dissolved in water, sterilized with 0.22 µm filters, and added to cultures at the indicated time and concentrations. Cell culture reagents were procured from GIBCO Life Technology (Grand Island, NY). Antibodies specific for the total and phosphorylated forms of JNK (p54/46) and p38 MAPK were obtained from Cell Signaling (Beverly, MA). Specific JNK inhibitor SP600125 was purchased from Calbiochem (La Jolla, CA). Fluorescent probes Fluo-3/AM and propidium iodide (PI) were procured from Molecular Probes (Eugene, OR). 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetra acetic acid tetrakis (acetoxy-methyl) ester (BAPTA-AM), ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetra acetic acid (EGTA), Hoechst 33258, and all other chemicals used in this study were obtained from Sigma and were of cell culture grade.

Cell culture. Macrophage cell line, J774A.1 (American Type Culture Collection TIB-67), established from BALB/c mouse, was maintained in Dulbecco's modified Eagle's medium, supplemented with 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin and 10% non-heat–inactivated fetal bovine serum (Atlanta Biologics, Atlanta, GA) in 5% CO2 atmosphere at 37°C. The J774A.1 cells were grown in 75 cm2 culture flasks and subcultured when the cells reached 70–80% confluence (every 3 days). Cells were used during the 3rd or 4th passages. Cultures were allowed to grow overnight (15 h) prior to the treatment. The concentrations used for various reagents, added 30 min prior to cadmium treatment, were 20 µM for SP 600125, 10 µM for BAPTA-AM, and 1 mM for EGTA. The used concentrations of the above agents were not cytotoxic.

Determination of intracellular Ca2+. [Ca2+]i levels were monitored by Fluo-3, which is a Ca2+-sensitive fluorescent indicator. Cells were seeded at 8 x 104 cells/well in 96-well microplates (Falcon, Becton Dickinson, Franklin Lakes, NJ) and treated with indicated time and concentration of cadmium. Cells were loaded with Fluo-3/AM (10 µM) in dark at 37°C for 1 h and washed twice with Tyrode's solution (137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 0.2 mM NaH2PO4, 12 mM NaHCO3, and 5.5 mM glucose). Cells were incubated in Tyrode's solution for another 30 min and the morphological fluorescence intensity of cells was determined using an Olympus IX71 inverted microscope (Olympus America, Melville, NY). A 488 nm excitation wavelength was used to illuminate Fluo-3, and fluorescence was detected at an emission wavelength of 510 nm. Digital images were acquired using the Magnafire SP (Olympus) digital camera.

Western blot analysis of phosphorylated JNK and p38 MAPK. The activation status (phosphorylation) of JNK and p38 MAPK was determined using phospho-specific antibodies as described previously (Johnson et al., 2003Go). Cells were grown at 2 x 106 cells/well in 6-well microplates and treated with cadmium for indicated time and concentrations. Following treatment, cells were washed with phosphate buffered saline (PBS), and total cell lysates were prepared by scraping in 100 µl of lysis buffer [20 mM Tris–HCl (pH 8.0), 1 mM sodium orthovanadate, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 2 mM ethylenediaminetetraacetate (EDTA), 1% Triton X-100, 50 mM ß-glycerolphosphate, and 10 µg/ml each of aprotinin, leupeptin, and pepstatin]. Fifty micrograms of proteins determined by Bradford assay was electrophoretically separated using a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to nitrocellulose paper followed by antibody staining. Equal loading and transfer of total protein was verified with the reversible Ponceau S stain (Sigma) dye and also by detecting total JNK and p38 MAPK. Immunodetection was performed using enhanced chemiluminescence (ECL) detection kit (Amersham Pharmacia, Piscataway, NJ).

Determination of caspase-3 activation. Caspase-3 activity was determined using CaspACE fluorometric activity assay (Promega, Madison, WI) with modifications as follows. Briefly, cells were treated in 96-well microplates after which Triton X-100 was added and repeatedly pipetted to lyse the cells. The homogenates were centrifuged at 4,000 x g for 10 min to remove cell debris. The supernatant was assayed for caspase-3 activities using CaspACE system according to the manufacturer's instructions. The plates were read at 360/460 nm (excitation/emission) using a SpectraMax Gemini plate reader (Molecular Devices, Sunnyvale, CA). The fluorescence signal was digitized and analyzed using SoftMax Pro (Molecular Devices, Irvine, CA).

Hoechst and PI 33258 staining. Apoptotic morphological changes in the nuclear chromatin of cells were detected by staining with the DNA binding fluorochrome Hoechst 33258 (bis-benzimide). Hoechst 33258 exhibits fluorescence enhancement upon binding to A-T rich regions of double-stranded DNA. Necrotic cell death was detected by staining with PI, a membrane impermeable dye excluded from viable cells. Propidium iodide binds to DNA by intercalating between the bases with little or no sequence preference and with a stoichiometry of one dye per 4–5 base pairs of DNA. Dead cells are PI-bright and live cells are PI-dim. Following 24 h treatment with cadmium, 120 µl of supernatant was removed and 20 µl of Hoechst 33258 (2 µg/ml) or PI (1 µg/ml) was added. The plates were read at 350/450 nm or 535/617 nm (excitation/emission) for Hoechst 33258 or PI fluorescence, respectively, using a SpectraMax Gemini plate reader. The fluorescence signal was digitized and analyzed using SoftMax Pro.

Terminal deoxynucleotidyl transferase (Tdt)-mediated dUTP nick end-labeling (TUNEL) assay. The TUNEL assay was performed using the in situ Cell Death Detection Kit (Roche Applied Science, Indianapolis, IN). Cells were plated at 8 x 104 cells/well in 96-well microplates and allowed to attach overnight. Cells were then treated with cadmium for 24 h, fixed with paraformaldehyde, and analyzed for stained nuclei according to the manufacturer's instructions. The fluorescence signal was read by a SpectraMax Gemini plate reader, digitized, and analyzed using SoftMax Pro. In addition, fluorescence microscopy was performed to examine fragmented DNA morphology using a IX71 inverted microscope. Digital images were captured using a MagnaFire SP Olympus digital camera.

Mitochondrial activity. MTT (3[4,5-dimethyl thiazolyl-2]2,5-diphenyl tetrazolium bromide, Sigma) assay was performed to investigate mitochondrial activity. Cells were seeded at 8 x 104 cells/well in 96-well microplates and treated with the indicated concentration of cadmium for 24 h. The cells were incubated with addition of 20 µl MTT (5 mg/ml). After 4 h, 120 µl of MTT media was taken from each well and 100 µl of 0.02 N HCl-isopropanol (warm) was added to dissolve formazan crystals. The absorbance of each cell was measure by UV spectrometer at 570 nm.

DNA synthesis as an index of proliferation. The [methyl-3H]thymidine incorporation assay was used as an index of proliferation. Cells were seeded at 8 x 104 cells/well in 96-well microplates. At 16 h prior to harvesting cells, each well was pulsed with 20 µl of [methyl-3H]thymidine (25 µCi/ml, 6.7 Ci/mmol, DuPont NEN Products, Boston, MA). Cells were harvested onto glass fiber filter paper (Cambridge Technology, Watertown, MA) using a cell harvester (PHD, Cambridge Technology). Proliferative response (uptake of [3H]thymidine) in the harvested cells was counted in a liquid scintillation counter (Pharmacia, Turku, Finland) and expressed as net disintegrations per minute (DPM).

Production of H2O2. The production of H2O2 was measured by detecting the fluorescent intensity of H2O2-sensitive probes after adding 5-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA, Molecular Probes). The cells were incubated in the presence of various concentrations of cadmium, and fluorescent intensity was recorded using a SpectraMax Gemini fluorescence plate reader. The CM-H2DCFDA fluorescence was detected by excitation at 485 nm, and emission at 530 nm. The fluorescence readings were digitized using SoftMax Pro. The results were similar in three independent replications, and data from a representative experiment (n = 5 wells) have been illustrated.

Replication and statistical analysis. Experiments were repeated at least 3–4 times with consistent results. Means ± SE from representative experiments have been presented. All statistical analyses were performed using the SAS statistical software (SAS Institute, Cary, NC). Treatment effects were analyzed using one-way analysis of variance (ANOVA) followed by Duncan's Multiple Range test. A p value of <0.05 was considered significant unless indicated otherwise in figure legends.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cadmium-Induced [Ca2+]i
To examine whether the [Ca2+]i change was involved in cytotoxicity by cadmium, we tested the [Ca2+]i using the calcium indicator Fluo-3. After cells were loaded with Fluo-3/AM, the fluorescent intensity of Fluo-3 was detected by fluorescence microscopy. A time-course study on elevated [Ca2+]i level was performed for 24 h with various cadmium concentrations (Fig. 1A). Cadmium at 20 and 500 µM showed distinct increase of [Ca2+]i at 2 h compared with the stable baseline level of [Ca2+]i in control cells. The changes in fluorescent intensity of peak calcium showed concentration-dependence. Cadmium at 20 µM slightly increased [Ca2+]i at 2 h, persisted for the whole period until 18 h, and this elevated [Ca2+]i tended to go down after that. Higher concentration of cadmium at 100 and 500 µM showed much stronger fluorescence intensity at earlier time point and the fluorescence declined away faster than that in lower concentration cells.



View larger version (86K):
[in this window]
[in a new window]
 
FIG. 1. Effect of CdCl2 on [Ca2+]i level observed by fluorescence microscopy. A. Cells were treated with 0, 20, 100 and 500 µM CdCl2 for 24 h. Altered [Ca2+]i levels were examined by Fluo-3, Ca2+-sensitive fluorescent indicator. Morphological fluorescence intensity of cell was visualized under a fluorescence microscope. For the closer look for altered [Ca2+]i levels, 2–3 cells are selected and magnified. B. Treated with 0 µM CdCl2 at 2 h. C. Treated with 500 µM CdCl2 at 2 h. The experiment was repeated three times with similar results and representative micrographs are shown.

 
Cadmium-Altered Activation of JNK and p38
We recently reported that cadmium induces activation of ERK in J774A.1 (Kim et al., 2003Go). To know whether cadmium alters activity of other MAPKs, including JNK and p38, phosphorylated forms of JNK and p38 were examined by Western blots. In J774A.1 cells treated with 20 µM cadmium, the levels of phosphorylated forms of p 54 (JNK2) and p46 (JNK1) increased clearly after 8 h (Fig. 2A). The phosphorylated form of JNK remained elevated even at 16 h and then declined at 24 h. In contrast, the levels of total (phosphorylation state-independent) JNK were not changed during the incubation period of 24 h. When cells were incubated with 1–50 µM cadmium for 16 h, the levels of phosphorylated JNK increased in a dose-dependent manner while those of total JNK were not changed (Fig. 2B). Besides activity of JNK changed by cadmium, altered p38 activity was also observed. After incubation with 20 µM cadmium for 16 h, the apparent bands of phosphorylated p38 started to decline (Fig. 3A). After 16 h of incubation, >20 µM cadmium resulted in suppression of p38 activity in a dose-dependent manner (Fig. 3B). However, the level of total p38 was not changed significantly, indicating that the specific kinase activity of p38 was down-regulated in cadmium-treated cultures.



View larger version (33K):
[in this window]
[in a new window]
 
FIG. 2. Effect of CdCl2 on phosphorylation of JNK. J774A.1 cells were exposed to CdCl2 at indicated concentrations and time. Cell extracts were analyzed by western blot to detect the p-JNK1 (p46) and p-JNK2 (p54) using a phosphospecific JNK antibody. P-JNK represents activated JNK whereas total JNK indicates total protein loading. A. JNK activation with CdCl2 20 µM over time. B. JNK activation after 16 h with different CdCl2 concentrations. Mean + SE (n = 3). *Indicates significant difference compared to control group at p < 0.05 analyzed using one-way ANOVA followed by Duncan's multiple range test.

 


View larger version (32K):
[in this window]
[in a new window]
 
FIG. 3. Effect of CdCl2 on phosphorylation of p38 MAPK. J774A.1 cells were exposed to CdCl2 at indicated concentrations and time. The level of total and p-p38 MAPK was measured by Western blot. P-p38 represents activated p38 whereas total p38 indicates total protein loading. A. p38 activation with CdCl2 20 µM over time. B. p38 activation after 16 h with different CdCl2 concentrations. Mean ± SE (n = 3). *Indicates significant difference compared to control group at p < 0.05 analyzed using one-way ANOVA followed by Duncan's multiple range test.

 
Role of Activated JNK on Cadmium-Induced Apoptosis and Necrosis
To examine the relationship between cadmium-induced JNK activation and apoptosis, we employed the JNK inhibitor anthrapyrazolone (SP600125; Bennett et al., 2001Go). J774A.1 cells were incubated with cadmium 20 µM in the absence or presence of 20 µM of SP600125 and assayed by caspase-3 activity, Hoechst 33258 staining, and TUNEL at 24 h. The levels of caspase-3 activity, Hoechst 33258 fluorescence, and TUNEL staining were similar in SP600125-treated or control cultures, but it significantly decreased in cultures treated with cadmium and SP600125 than in cultures treated with cadmium only (Fig. 4). Propidium iodide is used to distinguish cells that are in the late stage of apoptosis or necrosis, which lose plasma membrane integrity and are permeable to PI (Fig. 5). Inhibition of JNK activity by 20 µM SP600125 decreased fluorescence of PI staining, which was increased by 20 µM of cadmium at 24 h. We interpret the data to indicate that JNK activation is involved in cadmium-induced apoptosis and necrosis.



View larger version (28K):
[in this window]
[in a new window]
 
FIG. 4. Effect of SP600125 on CdCl2-induced caspase-3 activation and DNA fragmentation. J774A.1 cells were pre-treated with 20 µM SP600125 for 30 min and then exposed to CdCl2 (20 µM) for 24 h. A. The intensity of fluorescence on caspase-3 enzyme activity was measured. B. The intensity of fluorescence on apoptotic nuclei stained by Hoechst 33258 was read by microplate spectrofluorometer. C. The intensity of fluorescence on labeled DNA strand breaks by TUNEL-reaction after CdCl2 exposure was read by microplate spectrofluorometer. D. Fluorescence on TUNEL-positive nuclei was visualized under fluorescence microscopy. Mean ± SE (n = 3). Different letters on top of bars indicate a significant difference at p < 0.05 analyzed with ANOVA followed by Duncan's multiple range test.

 


View larger version (23K):
[in this window]
[in a new window]
 
FIG. 5. Effect of SP600125 on CdCl2-induced necrosis. J774A.1 cells were pre-treated with 20 µM SP600125 for 30 min and then exposed to CdCl2 (20 µM) for 24 h. The intensity of PI fluorescence after CdCl2 exposure was read by microplate spectrofluorometer. Mean ± SE (n = 3). Different letters on top of bars indicate a significant difference at p < 0.05 analyzed using ANOVA followed by Duncan's multiple range test.

 
Inhibitory Effect of BAPTA and EGTA on Cadmium-Induced [Ca2+]i Elevation, Growth Arrest, Mitochodrial Activity, and Necrosis
To verify the role of [Ca2+]i as a key second messenger, cells were pre-loaded with 10 µM BAPTA-AM and 1 mM EGTA for 30 min. BAPTA-AM is an effective membrane-permeable intracellular Ca2+ chelator, trapped in the cells after cytoplasmic hydrolysis. As shown in Figure 6A, chelating intracellular Ca2+ with BAPTA-AM prevented the elevation of [Ca2+]i, demonstrating that the release of intracellular Ca2+ is essential for cadmium-induced [Ca2+]i overloading. Extracellular Ca2+ removal by EGTA also diminished cadmium-induced [Ca2+]i overloading but showed slight elevation of [Ca2+]i, suggesting that the extracellular Ca2+ is an important source for elevated [Ca2+]i but that another source of intracellular Ca2+ storage is also important. Results presented in Figure 6B–C demonstrate that BAPTA-AM and EGTA pretreatment suppressed cadmium-induced growth arrest and mitochondrial impairment. BAPTA-AM and EGTA were able to reduce mitochondrial activity, indicating the importance of normal Ca2+ signaling on mitochondrial function. The inhibitory effect of [Ca2+]i chelation on cadmium-induced necrotic cell death examined by PI staining is shown in Figure 6D.



View larger version (37K):
[in this window]
[in a new window]
 
FIG. 6. Effect of BAPTA-AM and EGTA on CdCl2-induced [Ca2+]i elevation, growth arrest, mitochondrial activity impairment and necrosis. J774A.1 cells were pre-treated with BAPTA-AM (10 µM) or EGTA (1 mM) for 30 min and then exposed to CdCl2 (20 µM). A. Morphological fluorescence intensity of altered [Ca2+]i level was visualized under fluorescence microscope using Fluo-3 after 6 h CdCl2 exposure. Representative pictures from experiments that were replicated a minimum of three times are shown. B. Proliferation was measured by [3H]thymidine incorporation after 18 h CdCl2 exposure. C. Mitochondrial activity was measured by MTT assay after 24 h CdCl2 exposure. D. The intensity of PI fluorescence after 24 h CdCl2 exposure was read by microplate spectrofluorometer. Mean ± SE (n = 3). Different letters on top of bars indicate a significant difference at p < 0.05 analyzed using ANOVA followed by Duncan's multiple range test.

 
Inhibitory Effect of BAPTA-AM and EGTA on Cadmium-Induced Apoptosis
To determine the role of calcium in the regulation of cadmium-induced apoptosis, J774A.1 cells were incubated with 20 µM cadmium in the absence or presence of BAPTA-AM or EGTA. Treatment of cultures with cadmium resulted in activation of caspase-3 at 24 h. Chelating intracellular and extracelluar calcium totally inhibited cadmiun-induced caspase-3 activation (Fig 7A). The elevation of Hoechst 33258 fluorescence by cadmium was also abolished (Fig 7B). The TUNEL staining confirmed that over-loaded [Ca2+]i is an important mediator for cadmium-induced apoptotic cell death (Fig. 7C).



View larger version (30K):
[in this window]
[in a new window]
 
FIG. 7. Effect of BAPTA-AM and EGTA on CdCl2-induced caspase-3 activation and DNA fragmentation. J774A.1 cells were pre-treated with BAPTA-AM (10 µM) or EGTA (1 mM) for 30 min and then exposed to CdCl2 (20 µM). A. The intensity of fluorescence on caspase-3 enzyme activity was measured after treatment of cells with cadmium. B. The intensity of fluorescence on apoptotic nuclei stained by Hoechst 33258 (HO)–by microplate spectrofluorometer. C. The intensity of fluorescence on TUNEL positive nuclei–visualized under fluorescence microscope. Mean ± SE (n = 3). Different letters on top of bars indicate a significant difference at p < 0.05 analyzed with ANOVA followed by Duncan's multiple range test.

 
Inhibitory Effect of BAPTA-AM and EGTA on Cadmium-Altered MAPKs Activity
To delineate the further signaling pathways of elevated [Ca2+]i by cadmium, we examined the phosphorylation of MAPKs (JNK and p38) in J774A.1 cells. The immunoblot with phosphorylated form of JNK specific antibody revealed that BAPTA-AM and EGTA significantly, but not completely, inhibited cadmium-induced JNK activation (Fig. 8A). Downregulated p38 activity by cadmium was recovered up to control level in the presence of 10 µM BAPTA-AM or EGTA (Fig. 8B). A higher concentration of BAPTA-AM (50 µM) was not able to prevent p38 activity downregulated by cadmium.



View larger version (41K):
[in this window]
[in a new window]
 
FIG. 8. Effect of BAPTA-AM and EGTA on CdCl2-induced JNK activation and down-regulation of p38. J774A.1 cells were pretreated with BAPTA-AM (10 and 50 µM) or EGTA (1 mM) for 30 min and then exposed to CdCl2 (20 µM) for 16 h. A. Total and p-JNK. B. Total and p-p38 were measured by Western blot after 16 h CdCl2 exposure. Different letters on top of bars indicate a significant difference at p < 0.05 analyzed with ANOVA followed by Duncan's multiple range test (Mean ± SE, n = 3).

 
Relationship Between [Ca2+]i and ROS
H2DCFDA is a dye that specifically binds to H2O2. Analysis of cells stained with H2DCFDA revealed that 20 µM cadmium treatment caused significantly higher cellular level of H2O2 (Fig. 9). Chelating intracellular and extracellular calcium by BAPTA-AM and EGTA dramatically prevented cadmium-induced H2O2 generation, suggesting that cadmium-induced [Ca2+]i elevation may be critical for ROS generation.



View larger version (19K):
[in this window]
[in a new window]
 
FIG. 9. Interrelationship of ROS and [Ca2+]i elevation. Effect of BAPTA-AM and EGTA on CdCl2-induced H2O2. J774A.1 cells were pretreated with BAPTA-AM (10 µM) or EGTA (1 mM) for 30 min and then exposed to CdCl2 (20 µM) for 16 h. Fluorescence stained with H2DCFDA was read by microplate spectrofluorometer. Mean ± SE (n = 3). Different letters on top of bars indicate a significant difference at p < 0.05 analyzed with ANOVA followed by Duncan's multiple range test.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Results presented here suggest that cadmium-activated JNK, which plays a critical role in the apoptotic suicide of cells. Cadmium strongly stimulated JNK activity after 8 h exposure of J774A.1 murine macrophage cells, and this stimulation persisted for 16 h. The sustained JNK activation was Ca2+-dependent and served as a death signal in cadmium-induced apoptosis. Chelation of [Ca2+]i by BAPTA-AM and EGTA prevented the cadmium-induced H2O2 generation and hampered mitochondrial activity, JNK and caspase-3 activation, and apoptosis, confirming the early mediating role of Ca2+ during cadmium-induced apoptosis. We also present evidence that cadmium downregulates activation of p38 MAPK. Cadmium-mediated modulation of JNK and p38 MAPK activity was closely correlated with elevated [Ca2+]i. Chelating [Ca2+]i reduced H2O2 production, indicating that ROS act in concert with [Ca2+]i signaling.

The inhibitory effect of cadmium on intracellular mechanisms of Ca2+ regulation has been reported earlier. Cadmium inhibited Ca2+, extruding the Ca2+-ATPase pump in both endoplasmic reticulum and plasma membrane (Benters et al., 1996Go; 1997Go; Zhang et al., 1990Go). In isolated bovine liver nuclei, cadmium resulted in inhibition of ATP-dependent nuclear Ca2+ uptake (Hechtenberg and Beyersmann, 1994Go). Besides its inhibitory effect on the Ca2+-ATPase pump for sequestration of [Ca2+]i, cadmium is also known to disturb Ca2+ release from inositol 1,4,5-trisphosphate (IP3)-sensitive intracellular stores. Cadmium at 20 µM evoked a transient rise in cellular IP3, and the perturbation of the IP3/Ca2+ messenger system was suggested as an early and discrete effect of cadmium in E367 neuroblastoma cells (Benters et al., 1997Go). A specific cell surface metal ion receptor was suggested as an interacting site with cadmium in Xenopus oocytes to activate IP3-mediated Ca2+ release (Hague et al., 2000Go). Cadmium may interact with cell surface membrane proteins coupled to a pertussis toxin–sensitive G protein, which drives IP3 induction and Ca2+ release in primary murine macrophages (Misra et al., 2002Go). Inhibition of Ca2+ influx by chelating extracellular Ca2+ showed significantly reduced [Ca2+]i in the current study, but it was not totally abolished. This suggests that extracellular Ca2+ is required in [Ca2+]i elevation by cadmium; however, there are other sources of intracellular Ca2+ storage in response to cadmium damage.

We recently showed that cadmium induced activation of ERK in J774A.1 (Kim et al., 2003Go). In the present study, cadmium activated JNK and deactivated p38 MAPK in a concentration-dependent manner. In contrast to our finding of down-regulation of 38 MAPK, there have been reports showing that the cadmium-activated p38 MAPK is responsible for apoptosis, mitotic arrest, activation of heat shock factor 1, and induction of heat shock protein 70 (Chao and Yang, 2001Go; Galan et al., 2000Go; Hung et al., 1998Go). Deactivation of p38 MAPK has been shown to lead to both anti-apoptotic and pro-apoptotic responses. Exogenous nerve growth factor induced dephosphorylation of p38, which prevents Bcl-2 phosphorylation and apoptotic response in the lymphoblastoid CESS B cell line (Rosini et al., 2004Go). However, a natural anticancer depsipeptide, FR901228, induced apoptosis of ras-transformed 10T1/2 cells through suppression of p38 pathways (Fecteau et al., 2002Go). Our study indicates that a potential value of cadmium may involve aberrant regulation of Ras through suppression of the p38 MAPK pathway, leading to apoptosis in macrophages.

Disruption of Ca2+ homeostasis seems to take a part in initiating activation of MAPKs. Calcium was mobilized from intracellular stores by tributylin that played an important role for the phosphorylation of JNK and p38 MAPK in the CCRF-CEM human T-cell line (Yu et al., 2000Go). P38 MAPK was activated by cadmium in primary macrophages, and depletion of [Ca2+]i with BAPTA-AM inhibited such activation (Misra et al., 2002Go). We found the opposite phenomenon, i.e., downregulation on p38 MAPK; however, it is consistent with other studies in that Ca2+ is an important regulator of p38 MAPK activity. Recent evidence suggests that the JNK pathway may play an important role in triggering apoptosis and signaling with mitochondria. Activated JNK by thapsigargin was involved in loss of mitochondrial membrane potential and was downstream of caspase-3 in Jurket leukemia T cells (Srivastava et al., 1999Go). A rise in [Ca2+]i by thapsigargin promoted nitric oxide (NO) generation and induction of JNK activity and apoptosis, including activation of caspase-2 and caspase–9 in Jurkat T cells (Srivastava et al., 1999Go). This activity is consistent with our data showing that elevated-[Ca2+]i by cadmium-mediated generation of H2O2 and activation of JNK leading to caspase-3 activation and DNA fragmentation. Ca2+ and JNK must be an important regulator of immune cell death.

Cadmium exposure to J774A.1 exhibited increased oxidative stress induced by ROS and NO production, following single-strand breaks and apoptosis (Hassoun and Stohs., 1996Go). In general, inducing synthesis of protective sulfhydryl compounds, including metallothionein and glutathione, preceded lipid peroxidation and DNA damage in cadmium-treated cells; it also contributed to protection of cells from injury by cadmium (Beyersmann and Hechtenberg, 1997Go). In the present study, cadmium still was able to induce a level of H2O2 at 6 h, so the protective antioxidation system may be interrupted by cadmium damage. We have shown that the increase in H2O2 generation by cadmium was also detected in response to elevation of [Ca2+]i, suggesting a close relationship between Ca2+ and ROS in signal transduction pathways. Cadmium stimulates the proliferation of cultured mammalian cells only when applied at and below micromolar concentrations, whereas elevated concentrations and prolonged exposure induce cell inhibition (Beyersmann and Hechtenberg, 1997Go). We have shown that 20 µM of cadmium inhibits the proliferation of macrophages at 16 h, and buffering [Ca2+]i level with BAPTA-AM and EGTA was able to recover growth arrest (Fig. 6B). An earlier in vivo study found that cadmium exposure significantly decreased the number of thymocytes in S-phase, indicating the inhibitory effect of cadmium on DNA synthesis in these cells (Morselt et al., 1988Go). We also reported inhibited proliferation of J774A.1 macrophages by cadmium via G2/M arrest (Kim et al., 2003Go). However, the exact signaling pathways from [Ca2+]i elevation to growth arrest are largely unknown. Intracellular calcium elevation leading to oxidative stress and alterations in mitochondrial and nuclear function are thought to be major events in cadmium-mediated growth arrest in our study. Impairment of nuclear Ca2+ regulation caused by cadmium is suggested to provoke alterations in nuclear events related to gene expression and cell proliferation (Hechtenberg and Beyersmann, 1994Go). Interaction of cadmium with Ca2+-binding protein calmodulin was also suggested as a key factor in inhibition of cell proliferation (Powlin et al., 1997Go).

In summary, we have demonstrated here that cadmium-elevated [Ca2+]i level primarily from extracellular space, activated JNK, and downregulated p38. Caspase-3 activation was involved in apoptosis by cadmium. JNK was involved for cadmium-induced caspase-3 activation and apoptosis. Hydrogen peroxide generation was mediated by cadmium-induced [Ca2+]i. Rising [Ca2+]i concentration and ROS may cause Ca2+ influx into mitochondria and may disrupt normal metabolism of mitochondria, leading to apoptosis and growth arrest. Chelating [Ca2+]i recovered growth arrest, mitochondria impairment, and subsequently necrosis. Taken together, the findings of the current study indicate that cadmium-induced [Ca2+]i elevation triggers growth arrest, regulates phosphorylation/dephosphorylation of protein kinases, and modulates signal transduction pathways. JNK and caspase-3 activation by cadmium is interrelated with [Ca2+]i to modulate cadmium toxicity.


    ACKNOWLEDGMENTS
 
Partial support of this work provided by the Center for Academic Excellence in Toxicology at the University of Georgia and by the Fred C. Davison Endowment is gratefully acknowledged.


    NOTES
 

1 To whom correspondence should be addressed at Department of Physiology and Pharmacology, The University of Georgia, Athens, Georgia 30602-7389. Fax. (706) 542-3015. E-mail: rpsharma{at}vet.uga.edu


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Behra, R., and Gall, R. (1991). Calcium/calmodulin-dependent phosphorylation and the effect of cadmium in cultured fish cells. Comp Biochem. Physiol C. 100, 191–195.[CrossRef][ISI][Medline]

Bennett, B. L., Sasaki, D. T., Murray, B. W., O'Leary, E. C., Sakata, S. T., Xu, W., Leisten, J. C., Motiwala, A., Pierce, S., Satoh, Y. et al. (2001). SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase. Proc. Natl. Acad. Sci. U. S. A. 98, 13681–13686.[Abstract/Free Full Text]

Benters, J., Flogel, U., Schafer, T., Leibfritz, D., Hechtenberg, S., and Beyersmann, D. (1997). Study of the interactions of cadmium and zinc ions with cellular calcium homoeostasis using 19F-NMR spectroscopy. Biochem. J. 322 (Pt 3), 793–799.[ISI][Medline]

Benters, J., Schafer, T., Beyersmann, D., and Hechtenberg, S. (1996). Agonist-stimulated calcium transients in PC12 cells are affected differentially by cadmium and nickel. Cell Calcium 20, 441–446.[CrossRef][ISI][Medline]

Berridge, M. J., Lipp, P., and Bootman, M. D. (2000). The versatility and universality of calcium signalling. Nat. Rev. Mol. Cell Biol. 1, 11–21.[CrossRef][ISI][Medline]

Beyersmann, D., and Hechtenberg, S. (1997). Cadmium, gene regulation, and cellular signalling in mammalian cells. Toxicol. Appl. Pharmacol. 144, 247–261.[CrossRef][ISI][Medline]

Biagioli, M., Watjen, W., Beyersmann, D., Zoncu, R., Cappellini, C., Ragghianti, M., Cremisi, F., and Bucci, S. (2001). Cadmium-induced apoptosis in murine fibroblasts is suppressed by Bcl-2. Arch. Toxicol. 75, 313–320.[CrossRef][ISI][Medline]

Chacon, E., and Acosta, D. (1991). Mitochondrial regulation of superoxide by Ca2+: An alternate mechanism for the cardiotoxicity of doxorubicin. Toxicol. Appl. Pharmacol. 107, 117–128.[ISI][Medline]

Chao, J. I., and Yang, J. L. (2001). Opposite roles of ERK and p38 mitogen-activated protein kinases in cadmium-induced genotoxicity and mitotic arrest. Chem. Res. Toxicol. 14, 1193–1202.[CrossRef][ISI][Medline]

Crow, T., Xue-Bian, J. J., Siddiqi, V., and Neary, J. T. (2001). Serotonin activation of the ERK pathway in Hermissenda: Contribution of calcium-dependent protein kinase C. J. Neurochem. 78, 358–364.[CrossRef][ISI][Medline]

Descotes, J. (1992). Immunotoxicology of cadmium. IARC Sci. Publ. 385–390.

Ermak, G., and Davies K. J. A. (2001). Calcium and oxidative stress: from cell signaling to cell death. Mol. Immunol. 38, 713–721.[CrossRef][ISI]

Fecteau, K. A., Mei, J., and Wang, H. C. (2002). Differential modulation of signaling pathways and apoptosis of ras-transformed 10T1/2 cells by the depsipeptide FR901228. J. Pharmacol. Exp. Ther. 300, 890–899.[Abstract/Free Full Text]

Galan, A., Garcia-Bermejo, M. L., Troyano, A., Vilaboa, N. E., de Blas, E., Kazanietz, M. G., and Aller, P. (2000). Stimulation of p38 mitogen-activated protein kinase is an early regulatory event for the cadmium-induced apoptosis in human promonocytic cells. J. Biol. Chem. 275, 11418–11424.[Abstract/Free Full Text]

Hague, F., Matifat, F., Louvet, L., Brule, G., and Collin, T. (2000). The carcinogen Cd(2+) activates InsP3-mediated Ca2+ release through a specific metal ion receptor in Xenopus oocyte. Cell Signal. 12, 419–424.[CrossRef][ISI][Medline]

Hassoun, E. A., and Stohs, S. J. (1996). Cadmium-induced production of superoxide anion and nitric oxide, DNA single strand breaks and lactate dehydrogenase leakage in J774A.1 cell cultures. Toxicology 112, 219–226.[CrossRef][ISI][Medline]

Hechtenberg, S., and Beyersmann, D. (1994). Interference of cadmium with ATP-stimulated nuclear calcium uptake. Environ. Health Perspect. 102(Suppl 3), 265–267.

Hung, J. J., Cheng, T. J., Lai, Y. K., and Chang, M. D. (1998). Differential activation of p38 mitogen-activated protein kinase and extracellular signal-regulated protein kinases confers cadmium-induced HSP70 expression in 9L rat brain tumor cells. J. Biol. Chem. 273, 31924–31931.[Abstract/Free Full Text]

IARC (International Agency for Research on Cancer). (1993). Cadmium, mercury, and exposures in the glass manufacturing industry. Working group views and expert opinions. Monogr. Eval. Carcinog. Risks Hum. 58, 41–117.

Iryo, Y., Matsuoka, M., Wispriyono, B., Sugiura, T., and Igisu, H. (2000). Involvement of the extracellular signal-regulated protein kinase (ERK) pathway in the induction of apoptosis by cadmium chloride in CCRF-CEM cells. Biochem. Pharmacol. 60, 1875–1882.[CrossRef][ISI][Medline]

Jarup, L. (2003). Hazards of heavy metal contamination. Br. Med. Bull. 68, 167–182.[Abstract/Free Full Text]

Jarup, L., Berglund, M., Elinder, C. G., Nordberg, G., and Vahter, M. (1998). Health effects of cadmium exposure—A review of the literature and a risk estimate. Scand. J. Work Environ. Health 24(Suppl 1), 1–51.[ISI]

Johnson, V. J., He, Q., Kim, S. H., Kanti, A., and Sharma, R. P. (2003). Increased susceptibility of renal epithelial cells to TNF{alpha}-induced apoptosis following treatment with fumonisin B1. Chem. Biol. Interact. 145, 297–309.[CrossRef][ISI][Medline]

Kim, J., Kim, S., Johnson, V. J., and Sharma, R. P. (2003). Effect of cadmium on p53 and mitogen-activated protein kinases in a murine macrophage cell line: relation to apoptosis. Toxicol. Sci. 72S, 269 (abstract).

Lohmann, R. D., and Beyersmann, D. (1993). Cadmium and zinc mediated changes of the Ca2+-dependent endonuclease in apoptosis. Biochem. Biophys. Res. Commun. 190, 1097–1103.[CrossRef][ISI][Medline]

Long, G. J. (1997). Cadmium perturbs calcium homeostasis in rat osteosarcoma (ROS 17/2.8) cells; a possible role for protein kinase C. Toxicol. Lett. 91, 91–97.[CrossRef][ISI][Medline]

Matsuoka, M., and Igisu, H. (1998). Activation of c-Jun NH2-terminal kinase (JNK/SAPK) in LLC-PK1 cells by cadmium. Biochem. Biophys. Res. Commun. 251, 527–532.[CrossRef][ISI][Medline]

Misra, U. K., Gawdi, G., Akabani, G., and Pizzo, S. V. (2002). Cadmium-induced DNA synthesis and cell proliferation in macrophages: The role of intracellular calcium and signal transduction mechanisms. Cell Signal. 14, 327–340.[CrossRef][ISI][Medline]

Morselt, A. F., Leene, W., De Groot, C., Kipp, J. B., Evers, M., Roelofsen, A. M., and Bosch, K. S. (1988). Differences in immunological susceptibility to cadmium toxicity between two rat strains as demonstrated with cell biological methods. Effect of cadmium on DNA synthesis of thymus lymphocytes. Toxicology 48, 127–139.[CrossRef][ISI][Medline]

Powlin, S. S., Keng, P. C., and Miller, R. K. (1997). Toxicity of cadmium in human trophoblast cells (JAr choriocarcinoma): Role of calmodulin and the calmodulin inhibitor, zaldaride maleate. Toxicol. Appl. Pharmacol. 144, 225–234.[CrossRef][ISI][Medline]

Rosini, P., De Chiara, G., Bonini, P., Lucibello, M., Marcocci, M. E., Garaci, E., Cozzolino, F., and Torcia, M. (2004). Nerve growth factor–dependent survival of CESS B cell line is mediated by increased expression and decreased degradation of MAPK phosphatase 1. J. Biol. Chem. 279, 14016–14023.[Abstract/Free Full Text]

Schaeffer, H. J., and Weber, M. J. (1999). Mitogen-activated protein kinases: specific messages from ubiquitous messengers. Mol. Cell Biol. 19, 2435–2444.[Free Full Text]

Srivastava, R. K., Sollott, S. J., Khan, L., Hansford, R., Lakatta, E. G., and Longo, D. L. (1999). Bcl-2 and Bcl-XL block thapsigargin-induced nitric oxide generation, c-Jun NH2-terminal kinase activity, and apoptosis. Mol. Cell Biol. 19, 5659–5674.[Abstract/Free Full Text]

Stringaris, A. K., Geisenhainer, J., Bergmann, F., Balshusemann, C., Lee, U., Zysk, G., Mitchell, T. J., Keller, B. U., Kuhnt, U., Gerber, J., et al. (2002). Neurotoxicity of pneumolysin, a major pneumococcal virulence factor, involves calcium influx and depends on activation of p38 mitogen-activated protein kinase. Neurobiol. Dis. 11, 355–368.[CrossRef][ISI][Medline]

Thevenod, F., and Jones, S. W. (1992). Cadmium block of calcium current in frog sympathetic neurons. Biophys. J. 63, 162–168.[Abstract]

Weast, R. C., and Astle, M. J. (1982). Handbook of Chemistry and Physics. CRC Press, Inc., Boca Raton, FL.

Xu, C., Johnson, J. E., Singh, P. K., Jones, M. M., Yan, H., and Carter, C. E. (1996). In vivo studies of cadmium-induced apoptosis in testicular tissue of the rat and its modulation by a chelating agent. Toxicology 107, 1–8.[CrossRef][ISI][Medline]

Yan, L., and Cirillo, J. D. (2004). Infection of murine macrophage cell lines by Legionella pneumophila. FEMS Microbiol. Lett. 230, 147–152.[CrossRef][ISI][Medline]

Yu, Z. P., Matsuoka, M., Wispriyono, B., Iryo, Y., and Igisu, H. (2000). Activation of mitogen-activated protein kinases by tributyltin in CCRF-CEM cells: Role of intracellular Ca2+. Toxicol. Appl. Pharmacol. 168, 200–207.[CrossRef][ISI][Medline]

Zhang, G. H., Yamaguchi, M., Kimura, S., Higham, S., and Kraus-Friedmann, N. (1990). Effects of heavy metal on rat liver microsomal Ca2+-ATPase and Ca2+ sequestering. Relation to SH groups. J. Biol. Chem. 265, 2184–2189.[Abstract/Free Full Text]