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
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ABSTRACT |
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Key Words: cadmium; calcium; ROS; MAPKs; growth arrest; apoptosis.
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INTRODUCTION |
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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., 2000). 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, 1991
). Under oxidative stress, mitochondrial Ca2+ accumulation can switch from physiologically beneficial process to cell death signal (Ermak and Davies, 2001
). Ca2+-dependent processes induced by cadmium (<1 µM) activate p21ras-dependent MAPK pathways, and nuclear factor-
B (NF-
B)dependent gene expression, to stimulate proliferation in peritoneal macrophages (Misra et al., 2002
). 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, 1982). Cadmium is a potent Ca2+ channel blocker and inhibits Ca2+ cellular uptake (Thevenod and Jones, 1992
). Cadmium has a high affinity for and activates calmodulin, a Ca2+-binding protein that regulates a variety enzymes and cell progresses (Behra and Gall., 1991
). 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, 1993
; Long, 1997
; Misra et al., 2002
).
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 signalrelated kinase (ERK) (Schaeffer and Weber, 1999). 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, 1999
). We recently reported ERK signalingdependent G2/M arrest and cell death in murine macrophages by cadmium (Kim et al., 2003
). 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, 1998
). Calcium-dependent PKC activation by serotonin contributed to ERK phosphorylation in the nudibranch mollusk Hermissenda (Crow et al., 2001
). Elevated [Ca2+]i was involved in p38 MAPK-induced neuronal cell death by pneumolysin (Stringaris et al., 2002
). 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, 1992). 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, 2004
). 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.
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MATERIALS AND METHODS |
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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-heatinactivated 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 7080% 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., 2003). 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 TrisHCl (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 45 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 34 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.
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RESULTS |
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DISCUSSION |
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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., 1996; 1997
; Zhang et al., 1990
). In isolated bovine liver nuclei, cadmium resulted in inhibition of ATP-dependent nuclear Ca2+ uptake (Hechtenberg and Beyersmann, 1994
). 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., 1997
). 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., 2000
). Cadmium may interact with cell surface membrane proteins coupled to a pertussis toxinsensitive G protein, which drives IP3 induction and Ca2+ release in primary murine macrophages (Misra et al., 2002
). 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., 2003). 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, 2001
; Galan et al., 2000
; Hung et al., 1998
). 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., 2004
). However, a natural anticancer depsipeptide, FR901228, induced apoptosis of ras-transformed 10T1/2 cells through suppression of p38 pathways (Fecteau et al., 2002
). 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., 2000). P38 MAPK was activated by cadmium in primary macrophages, and depletion of [Ca2+]i with BAPTA-AM inhibited such activation (Misra et al., 2002
). 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., 1999
). 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 caspase9 in Jurkat T cells (Srivastava et al., 1999
). 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., 1996). 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, 1997
). 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, 1997
). 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., 1988
). We also reported inhibited proliferation of J774A.1 macrophages by cadmium via G2/M arrest (Kim et al., 2003
). 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, 1994
). Interaction of cadmium with Ca2+-binding protein calmodulin was also suggested as a key factor in inhibition of cell proliferation (Powlin et al., 1997
).
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.
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ACKNOWLEDGMENTS |
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NOTES |
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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
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