Abstract
Metallothioneins (MTs) are cytoplasmic proteins that sequester certain divalent cations and are considered a primary cellular defense against the toxic transition metal cadmium (Cd2+). MT-I/II(–/–) knockout [MT(–/–)] cells are available and serve as an excellent tool to study non–MT-related mechanisms in metal tolerance. In the current study, Cd2+-resistant MT(–/–) (CdR) and CdR revertant (CdR-rev) cell lines were developed and characterized to investigate non–MT-mediated cellular protection mechanisms. Resistance to Cd2+ was approximately 70-fold higher in CdR than the parental MT(–/–) cell line (IC50 = 20 versus 0.3 μM, respectively) and was stable in the absence of Cd2+ for 35 days. Accumulation of Cd2+ by the CdR cell line was reduced by approximately 95% compared with parental cells, primarily because of a decreased Cd2+ uptake. Cd2+ uptake by the MT(–/–) parental cell line was independent of sodium, energy, and electrogenic potential. Uptake was saturable (Km = 65 nM; Vmax = 4.9 pmol/mg/min) and pH-dependent (maximal at pH 6.5–7). Potent inhibitors of Cd2+ uptake included Zn2+ (IC50 = 7 μM), Mn2+ (IC50 = 0.4 μM), and the T-type Ca2+ channel antagonist mibefradil (IC50 = 5 μM), whereas other metals (including Fe2+) and L-type Ca2+ channel antagonists had little effect. Immunoblot and real-time reverse transcription-polymerase chain reaction analysis indicated that the Cacnα1G T-type Ca2+ channel was expressed at a reduced level in CdR compared with the parental MT(–/–) cell line, suggesting it is important for Cd2+ uptake. The CdR1-rev cell line was found to have a Cd2+ uptake and sensitivity level in between that of the CdR1 and MT(–/–) cell lines. Consistent with this was an intermediate expression of Cacnα1G in the CdR-rev cell line. These data suggest that decreased expression of Cacnα1G protects cells from Cd2+ exposure by limiting Cd2+ uptake.
The heavy metal cadmium (Cd2+) is naturally present in soil, sediment, air, and water and has been concentrated in the human environment through its industrial use. Cd2+ exposure leads to a variety of adverse health effects, including osteoporosis, nonhypertrophic emphysema, irreversible renal tubular injury, and anemia (Waisberg et al., 2003). In addition, Cd2+ is a human carcinogen, and occupational exposure has been associated with cancers of the lung and possibly prostate, pancreas, and kidney (Waalkes, 2003). The main nonoccupational human exposure to Cd2+ is from cigarette smoke, whereas for nonsmokers the diet is the predominant source (Satarug and Moore, 2004).
The metallothioneins (MTs) are a family of low-molecular-weight (6–7 kDa), thiol-rich, metal binding proteins (Klaassen et al., 1999). All vertebrates express two or more distinct MT isoforms designated MT I through MT IV (Palmiter, 1998). In mammals, MT I and MT II are ubiquitously expressed in all tissues and are considered the major forms (Palmiter, 1998). Cell lines and transgenic mice deficient in MT are sensitive to Cd2+, whereas mice and cells overexpressing MT I and MT II are resistant (Klaassen et al., 1999). In addition, selection of cultured mammalian cells in Cd2+ typically results in resistance based on multiple amplifications of the entire MT locus (Palmiter, 1998). MT can bind large amounts of Cd2+ and is considered the predominant cellular defense mechanism against Cd2+ toxicity (Klaassen et al., 1999).
The level of MT gene expression is impacted by a variety of circumstances and differs widely with factors such as tissue type, cell type, gender, and pathological state (Waalkes and Pérez-Ollé, 2000). In addition, remarkable interindividual variation in MT expression level in human tissues such as liver, kidney, and red blood cells has been reported previously (Onosaka et al., 1985; Bem et al., 1988; Silevis-Smitt et al., 1992; Yoshida et al., 1998; Allan et al., 2000; Wu et al., 2000). Thus, it is important to understand potential non–MT-mediated cellular protection pathways, because these could be important alternative or auxiliary mechanisms for persons who have relatively poor MT expression. Altered cellular transport characteristics could reduce accumulation of Cd2+, through either increased efflux or decreased uptake, and could be important for tissue protection. Such mechanisms have not been extensively characterized at the molecular level. A large body of literature exists describing the importance of glutathione-dependent biliary excretion of metals, including Cd2+, in vivo (for review, see Leslie et al., 2005). Studies in rats deficient in Mrp2 (Abcc2), an ATP-binding cassette transporter protein involved in the cellular efflux of many xenobiotics, implicate Mrp2 as the major canalicular membrane transporter involved in biliary excretion of Cd2+ (Dijkstra et al., 1996; Paulusma and Oude Elferink, 1997). A candidate transporter for cellular uptake of Cd2+ is the divalent metal transporter (Dmt1/Slc11a2), also known as the natural resistance-associated macrophage protein 2 or divalent cation transporter 1, a proton-coupled metal-ion transporter (Gunshin et al., 1997). Several in vitro studies have firmly established human DMT1/SLC11A2 as a transporter of Cd2+ (Bressler et al., 2004). In addition, a variety of studies have suggested that Cd2+ transport into cells occurs through transporters and ion channels involved in the passage of physiological metals such as Zn2+, Mn2+, and Ca2+ (Waalkes and Poirier, 1985; Friedman and Gesek, 1994; Souza et al., 1997; Yanagiya et al., 2000; Zalups and Ahmad, 2003).
Except for the recently identified solute carrier protein ZIP8/Slc39a8 (Dalton et al., 2005), transporters and channels that Cd2+ could gain cellular entry through “molecular mimicry” have not been specifically identified at the molecular level. For example, high concentrations of Ca2+ channel antagonists have been shown to inhibit cellular uptake of Cd2+ (Friedman and Gesek, 1994; Souza et al., 1997). However, concentrations of antagonists used in these studies could inhibit several classes of Ca2+ and/or other channel and transport proteins. By using an MT-I/II(–/–) knockout [MT(–/–)] cell line made resistant to Cd2+ by continuous exposure in a stepwise manner (Yanagiya et al., 1999), potential non–MT-mediated cellular protection mechanisms from Cd2+ have been studied (Yanagiya et al., 1999, 2000; Himeno, 2002; Himeno et al., 2002). The Cd2+-resistant MT(–/–) cell line had a reduced ability to take up manganese (Mn2+), and it was concluded that the MT(–/–) cell line expresses a novel Mn2+ transport system by which Cd2+ can enter mammalian cells (Yanagiya et al., 2000). However, such a transporter is yet to be identified.
In the present work, a Cd2+-resistant MT(–/–) murine fibroblast cell line (CdR) and a revertant cell line (CdR-rev) have been established and Cd2+ transport extensively characterized. Resistance was predominantly because of a reduced Cd2+ uptake, probably through a specific decrease in expression of the T-type Ca2+ channel Cacnα1G, suggesting that Cacnα1G could be an important pathway for Cd2+ entry into cells.
Materials and Methods
Materials. [109Cd]CdCl2 (3 mCi/mg) was purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). Verapamil, mibefradil, diltiazem, nicardipine, rotenone, sodium azide, choline, MnCl2 (Mn2+), ZnCl2 (Zn2+), NiCl2 (Ni2+), Pb(CH3COO)2 (Pb2+), FeSO4 (Fe2+), CuCl2 (Cu2+), AlCl3 (Al3+), HgCl2 (Hg2+), NaAsO2 (As3+), and CdCl2 (Cd2+) were purchased from Sigma-Aldrich (St. Louis, MO).
Establishment of Cd2+-Resistant and -Revertant Cell Lines. Previously established simian virus 40-transformed MT(–/–) and MT(+/+) fibroblast cells that originated from whole embryonic cells (Kondo et al., 1999) were cultured in high-glucose DMEM with 10% fetal bovine serum under 5% CO2 at 37°C. CdR cells were developed by continuous exposure to increasing concentrations of Cd2+ up to a final concentration of 10 μM, according to the method of Yanagiya et al. (1999). Cells were cultured for several weeks in the presence of 10 μM Cd2+ and then cloned by limiting dilution. Cloned cells (CdR1 and CdR2) were grown in the absence of Cd2+ (4–10 days) before use in experiments. Revertant cell lines (CdR1-rev and CdR2-rev) were established by growing CdR1 and CdR2 in the absence of Cd2+ for up to 238 days.
Cytotoxicity Testing. MT(–/–), MT(+/+), CdR1, CdR2, CdR1-rev, and CdR2-rev cell lines were plated at 1 × 104 cells/well in a 96-well plate, and 24 h later they were treated with Cd2+ (0.1–300 μM) for 72 h. The tetrazolium-based microtiter plate assay CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI), was then used to measure acute cytotoxicity, according to the manufacturer's instructions. Results are expressed as a percentage of untreated control.
Cd2+Total Accumulation, Efflux, and Uptake by MT(–/–), CdR, and CdR1-rev Cells. Cells were plated at 2 × 105 cells/well in six-well plates and cultured 24 h before treatment with carrier-free 109Cd. For total accumulation studies, cells were incubated with 109Cd (0.1, 0.3, and 1 μM; 40–400 nCi) for 24 h. Cells were then harvested by washing three times with 2 ml of ice-cold PBS containing 0.05% (w/w) EDTA and lysed with PBS containing 2% SDS. Protein content was measured using the DC Protein Assay kit (Bio-Rad, Hercules, CA), and radioactivity was normalized per milligram of protein. For measurement of cellular Cd2+ efflux, cells were incubated for 6 h with 109Cd (0.1 μM; 40 nCi) in serum-free media, cells were then washed three times with PBS containing 0.05% EDTA (37°C), incubated at 37°C in serum-free media for the indicated time points, and harvested as described above for total accumulation. For uptake studies, cells were incubated with 109Cd (0.1 μM) in serum-free media for 1 h or at the indicated time points and harvested as described above for total accumulation.
Sodium dependence of uptake was measured using transport buffer (137 mM NaCl replaced with equimolar choline chloride or LiCl, 5.33 mM KCl, 1.8 mM CaCl2, 0.814 mM MgCl2, 10 mM HEPES, and 25 mM glucose). The effect of the metabolic inhibitors sodium azide (10 mM) and rotenone (10, 30, and 100 μM) on 109Cd uptake was assessed in glucose-free DMEM with the addition of mannitol (25 mM) to maintain osmolarity. Cells were preincubated for 1 h with these inhibitors before the addition of 109Cd and measurement of uptake. The effects of other potential modulators of 109Cd uptake [Zn2+ (0–30 μM), Mn2+ (0–30 μM), Ni2+ (0–300 μM), Fe2+ (0–100 μM), Pb2+ (0–100 μM), pH change (pH 5, 6, 6.5, 7, 7.5, or 8), and the Ca2+ channel antagonists mibefradil (0–30 μM), verapamil (0–100 μM), diltiazem (0–300 μM), and nicardipine (0–100 μM)] were evaluated, without preincubation of modulators, in serum-free DMEM.
Kinetic parameters of uptake were determined by measuring the initial rate of 109Cd uptake at eight different substrate concentrations (3–300 nM) at a single time point of 1 h. Kinetic parameters were determined using nonlinear regression analysis (Prism; Graph-Pad Software Inc., San Diego, CA).
Real-Time RT-PCR Analysis. Gene expression was quantified using real-time RT-PCR analysis as described previously (Walker, 2001). In brief, total RNA was isolated from cell lines using an RNeasy midi kit (QIAGEN, Valencia, CA) according to the manufacturer's instructions, reverse transcribed with murine leukemia virus reverse transcriptase and oligo(dT) primers. The forward and reverse primers for selected genes were designed using Primer Express software (Applied Biosystems, Foster City, CA) and are shown in Table 1. The SYBR Green DNA polymerase chain reaction kit (Applied Biosystems) and the MyIQ single-color real-time polymerase chain reaction detection instrument (Bio-Rad) were used for real-time RT-PCR analysis. The relative differences in expression between groups were expressed using cycle time (Ct) values, and the Ct values for genes of interest were first normalized with that of β-actin in the same sample, and then differences between groups were expressed relative to controls set as 100%. Assuming that the Ct value is reflective of the starting copy number and there is 100% efficiency, a difference of one cycle is equivalent to a 2-fold difference in starting copy number using the 2(–dCt) formula. Real-time RT-PCR was performed in triplicate, and similar results were obtained from standard curves produced for each gene analyzed.
Immunoblot Analysis. Cell homogenates were prepared, and relative levels of Cacnα1G were determined by immunoblot analysis essentially as described previously (Yunker et al., 2003). In brief, cells were washed twice with ice-cold PBS and then collected with a plastic cell scraper in 1 ml of HEPES (50 mM; pH 7.4) buffer containing EGTA (1 mM) and protease inhibitors (Complete cocktail tablets; Roche Diagnostics, Indianapolis, IN). Cell homogenates (50 μg) and a positive control homogenate prepared from murine cerebellum (75 μg) were subjected to SDS-polyacrylamide gel electrophoresis and electrotransferred to a nylon membrane. Blots were blocked in 5% (w/v) skim milk powder for 1 h followed by incubation with the CW53 Cav3.1/Cacnα1G antibody in 3% (w/v) bovine serum albumin overnight at 4°C. After washing, blots were incubated with horseradish peroxidase-conjugated donkey anti-rabbit antibody (GE Healthcare, Little Chalfont, Buckinghamshire, UK) followed by application of chemiluminescence blotting substrate (SuperSignal Dura Extended Duration; Pierce Chemical, Rockford, IL).
Results
Establishment of a Cd2+-Resistant Cell Line. A CdR variant of the murine fibroblast MT(–/–) cell line was established by culturing these cells in gradually increasing concentrations of Cd2+ up to 10 μM. After establishment and continuous culture in 10 μM Cd2+ for 10 weeks, two clones (CdR1 and CdR2) were produced that had similar resistance levels to Cd2+ (IC50 of ∼20 μM), approximately 70- and 7-fold more resistant than the sensitive MT(–/–) parental cell line (IC50 = 0.3 μM) and the MT(+/+) cell line (IC50 = 3 μM), respectively (Fig. 1). To evaluate the stability of the CdR phenotype, the CdR1 cell line was also grown in the absence of Cd2+, and the resistance to Cd2+ was evaluated over time (Table 2). Resistance was stable for a minimum of 35 days without Cd2+ exposure. At 63 days of culture in the absence of Cd2+, resistance had decreased 40% with an IC50 of 9 μM versus 15 μM for cells continuously cultured in the presence of Cd2+. After 238 days of culture in the absence of Cd2+, resistance had decreased by 96% (IC50 = 0.8 versus 18 μM for absence and presence of Cd2+, respectively), and this was considered the revertant cell line.
Resistance Is Mediated Primarily through Reduced Cd2+Uptake. To discern the mechanism of resistance to Cd2+, the total accumulation of 109Cd over 24 h was measured in MT(–/–) parental, CdR1, and CdR2 cell lines (Fig. 2A). CdR1 and CdR2 cell lines accumulated approximately 95% less Cd2+ than the MT(–/–) parental cell line over Cd2+ concentrations of 0.1, 0.3, and 1 μM. Further experiments revealed that uptake of Cd2+ (0.1 μM; 40 nCi) by CdR1 and CdR2 cell lines was reduced by approximately 58, 70, 85, and 92% compared with the MT(–/–) parental cell line at 15, 30, 60, and 120 min, respectively (Fig. 2B). Over 30 min, efflux of Cd2+ from the MT(–/–) parental cell line was not detected; however, approximately 40% of Cd2+ was effluxed from CdR1 and CdR2 cell lines at 1 min at which point a plateau was reached (Fig. 2C). Although increased efflux does occur in the CdR clones compared with the sensitive parental MT(–/–) cell line, initial Cd2+ accumulation was only approximately 5% in the CdR1 and CdR2 cells compared with the parental cell line because of decreased uptake (Fig. 2C). Despite the lack of MT, once Cd2+ had entered the MT(–/–) parental cell line it was accumulated (Fig. 2, A–C). This could be because of the lack of an efflux mechanism for Cd2+ in this cell line and/or a non-MT mechanism for intracellular sequestration. Overall, these data suggest that a decreased uptake was the primary mechanism by which CdR1 and CdR2 acquired resistance to Cd2+.
Cd2+Uptake Is Independent of Sodium, Energy, and Electrogenic Potential. Because a large part of the CdR phenotype seemed to be conferred by a decreased uptake of Cd2+ into CdR1 and CdR2 cell lines, Cd2+ influx was characterized using the MT(–/–) parental cell line. When NaCl was substituted with choline or LiCl in transport buffer, cellular Cd2+ uptake was increased or unchanged, respectively, indicating that this is not a Na+-dependent process (Fig. 3A). When cells were depolarized with 10-fold higher external K+(54 mM) than control (5.4 mM) concentrations, little difference in Cd2+ uptake was observed, indicating that this process is not dependent on the electrogenic potential of the cell (Fig. 3B). An energy requirement for uptake was evaluated using the metabolic inhibitors sodium azide (10 mM) and rotenone (10, 30, and 100 μM) (Fig. 3C). These inhibitors had no significant effect on Cd2+ uptake.
Kinetic Analysis of Cd2+Uptake. Cd2+ uptake was further characterized by determining initial rates of uptake with several concentrations of Cd2+ (Fig. 4A). Uptake was saturable and according to nonlinear regression analysis, the apparent Km for Cd2+ was 65 ± 0.32 nM, and the Vmax was 4.9 ± 0.1 pmol mg–1 min–1. These kinetic parameters are consistent with values reported previously (Yanagiya et al., 2000).
Cd2+Uptake Is pH-Dependent. To determine whether uptake of Cd2+ by the MT(–/–) parental cell line was influenced by pH, transport was measured over a pH range of 5 to 8. Uptake of Cd2+ was minimal at pH 5 to 6 (0.3 pmol mg–1 min–1), maximal between pH 6.5 and 7 (6.5 pmol mg–1 min–1), and decreased to 1.6 pmol mg–1 min–1 between pH 7.5 and 8 (Fig. 4B). Uptake of Cd2+ by the CdR1 cell line was minimal over the pH range examined (Fig. 4B).
Cd2+Uptake Is Modified by Several Metals. The ability of several transition metals and metalloids to alter the uptake of Cd2+ was evaluated. As a preliminary experiment, the effect of a single concentration (10 μM) of Pb2+, Cu2+, Fe2+, Al3+, Ni2+, Zn2+, Mn2+, Hg2+, and As3+ on Cd2+ (0.1 μM; 40 nCi) uptake was quantified (Fig. 5A). At this concentration, Fe2+, Al3+, Ni2+, Hg2+, and As3+ had no effect on Cd2+ uptake. Several compounds were selected for further characterization through generation of concentration-response curves and IC50 value determination (Fig. 5, B–F).
Consistent with the results of Yanagiya et al. (2000), Zn2+ and Mn2+ were found to be potent inhibitors of Cd2+ uptake with IC50 values of 7 and 0.4 μM, respectively (Fig. 5, B and C). Although Fe2+ had no effect on Cd2+ uptake at 10 μM, it was tested at increasing concentrations because the Fe2+ uptake transporter Dmt1/Slc11a2 is known to be important for Cd2+ uptake in other cell systems (Bressler et al., 2004). However, Fe2+ did not inhibit the uptake of Cd2+ at the concentrations tested (up to 100 μM) (Fig. 5D). Pb2+ had an unexpected stimulatory effect on Cd2+ uptake with an activity 155% of control at 100 μM (Fig. 5E). Although Ni2+ had little effect at 10 μM (Fig. 5A), it was further characterized at increasing concentrations because of previous reports that Ni2+ can inhibit certain Ca2+ channels, which are possibly involved in cellular translocation of Cd2+ (Lacinová et al., 2000). Ni2+ was not a potent inhibitor of Cd2+ uptake with an IC50 value of 300 μM (Fig. 5F).
Cd2+Uptake Is Altered by Several Ca2+Channel Antagonists. A series of Ca2+ channel antagonists were tested for their ability to inhibit Cd2+ uptake by the parental MT(–/–) cell line. Mibefradil, which has been shown to be a relatively specific inhibitor of T-type Ca2+ channels, proved to be a potent inhibitor of Cd2+ uptake by the parental MT(–/–) cell line with an IC50 value of 5 μM (Fig. 6A). The phenylalkylamine verapamil, an L-type Ca2+ channel antagonist, inhibited Cd2+ uptake with an IC50 value of 60 μM (Fig. 6B). Diltiazem and nicardipine are drugs from different chemical classes of L-type Ca2+ channel antagonists, the benzothiazepines and dihydropyridines, respectively. Neither diltiazem (IC50 > 300 μM) nor nicardipine (IC50 > 100 μM) was a potent inhibitor of Cd2+ uptake (Fig. 6, C and D). The overall pattern of inhibition by these Ca2+ channel antagonists indicates that a T-type Ca2+ channel is involved in Cd2+ uptake.
CdR1-rev Shows Intermediary Cd2+Uptake and Sensitivity. As a tool to identify changes of functional relevance for the Cd2+ resistance in CdR cells, a CdR1-rev cell line was developed by growing the CdR1 cell line in the absence of Cd2+ for 238 days. The CdR1-rev was compared with the MT(–/–) parental and CdR1 cell lines for the following: Cd2+ cytotoxicity (Fig. 7A; Table 2); Cd2+ efflux (Fig. 7B); Cd2+ uptake (Fig. 7C); and the impact of Mn2+, Zn2+, and the Ca2+ channel antagonist mibefradil on Cd2+ uptake (Fig. 7D). The CdR1-rev cell line was of intermediate sensitivity to Cd2+ cytotoxicity (IC50 = 0.8 μM) compared with the CdR1 (IC50 = 18 μM) and MT(–/–) parental (IC50 = 0.2 μM) cell lines (Fig. 7A). Cd2+ efflux from the CdR1-rev cell line was not significantly different from that of the MT(–/–) parental cell line, suggesting that efflux is not the predominant protective mechanism (Fig. 7B). Consistent with the sensitivity to Cd2+, uptake by the CdR1-rev cell line was intermediate between the CdR1 and MT(–/–) parental cell lines. Uptake in CdR1-rev cells was approximately 50% higher than that of CdR1 and 50% lower than that of MT(–/–) parental cell lines (Fig. 7C). Mn2+,Zn2+, and mibefradil inhibited Cd2+ uptake by the CdR1-rev cell line by 44, 65, and 38%, respectively, relative to vehicle control (Fig. 7D). Cd2+ uptake by the MT(–/–)-sensitive parental cell line was inhibited to a similar extent (relative to vehicle control) by Mn2+, Zn2+, and mibefradil (66, 74, and 47%, respectively). As expected, the uptake of Cd2+ by the CdR1 cell line was not affected by Mn2+ or mibefradil, whereas it was inhibited modestly by Zn2+(31% inhibition) (Fig. 7D).
Expression Levels of the T-Type Ca2+ Channel Cacnα1G Suggest Potential Importance for Cd2+ Uptake. The relative expression levels of the Ca2+ channels Cacnα1G and Cacnβ3 were found to be decreased in CdR clones compared with the MT(–/–) parental cell line in preliminary microarray analysis [further genomic analysis will be reported in a separate article (E. M. Leslie, J. Liu, D. M. K. Ducharme, and M. P. Waalkes, manuscript in preparation)]. This prompted further characterization of Cacnα1G and Cacnβ3 expression in the CdR1-rev, CdR1, and MT(–/–) parental cell lines using real-time RT-PCR and immunoblot analysis (Fig. 8). Consistent with the intermediate function of the CdR1-rev cell line, the Cacnα1G T-type Ca2+ channel was expressed at a level between that of the CdR1 and MT(–/–) parental cell lines. Thus, the Cacnα1G T-type Ca2+ channel was expressed in CdR1 cells at 0.2% of the expression in the MT(–/–) parental cell line and at approximately 8% of the CdR1-rev (Fig. 8A). The protein expression of Cacnα1G was also analyzed and confirmed the RNA expression level differences between the cell lines (Fig. 8B). Protein expression was not detectable in cell homogenate prepared from the CdR1 cell line, a faint band was visible in the CdR1-rev, whereas the MT(–/–) parental cell line contained a bright band at a molecular mass of ∼240 kDa, consistent with the murine cerebellum-positive control. The CW53 antibody had been rigorously tested for Cacnα1G specificity in a previous study (Yunker et al., 2003). The other Ca2+ channel subunit, Cacnβ3 (Fig. 8C), did not differ in expression levels between the CdR1 and CdR1-rev cell lines, indicating that this protein is unlikely to influence Cd2+ uptake.
Expression Levels of Dmt1/Slc11a2, AE4/Slc39a7, and Zip8/Slc39a8 Are Unchanged. The expression levels of two Mn2+ transporters, Dmt1/Slc11a2 and AE4/Slc39a7, were measured because of the proposed importance of Mn2+ transporters in Cd2+ uptake. Dmt1/Slc11a2 mRNA levels were not significantly different in the MT(–/–) cell line compared with the CdR1-rev or the CdR1 cell lines (Fig. 9A). AE4/Slc39a7 has been proposed to transport Mn2+ (Eide, 2004); however, expression was similar in the CdR1 versus MT(–/–) parental cell line (Fig. 9B) and therefore unlikely to be involved in the Cd2+ resistance observed. The expression of ZIP8/Slc39a8, a solute carrier protein that has recently been shown to facilitate uptake of Cd2+ in transfected cell lines and is thought to be associated with Cd2+ sensitivity (Dalton et al., 2005), was similar in the CdR1 versus the MT(–/–) parental cell line (Fig. 9C). Therefore, ZIP8/Slc39a8 is unlikely to be involved in the differential Cd2+ uptake and sensitivity in the CdR and MT(–/–) parental cell lines.
Expression of the Aquaporin 1 Channel Is Associated with Increased Cd2+Uptake. In addition to changes in Ca2+ channel expression levels, microarray analysis indicated that expression of the zinc-iron regulated transporter-like (Zirtl/Slc39a1) and aquaporin 1 were decreased in the CdR1 and CdR2 clones compared with the MT(–/–) parental cell line (E. M. Leslie, J. Liu, D. M. K. Ducharme, and M. P. Waalkes, manuscript in preparation). This was confirmed by real-time RT-PCR analysis (Fig. 9, D and E). However, the expression of Zirtl/Slc39a1 was not significantly different in the CdR1 and CdR1-rev cell lines, suggesting that this transporter is not related to the observed functional changes in Cd2+ toxicokinetics. Aquaporin 1, an integral membrane protein that serves as a channel for the transfer of water across the cell membrane, was expressed in the CdR1 clone at 22% of the level found in the parental MT(–/–) cell line (Fig. 9E). Furthermore, the CdR1-rev cell line expressed aquaporin 1 at an intermediate level between the MT–/– parental (1.4-fold lower) and CdR1 (3.3-fold higher) cell lines (Fig. 9E), consistent with the intermediate phenotype of the revertant cell line (Fig. 7).
Discussion
In this work, a Cd2+-resistant MT(–/–) cell line was established and characterized to investigate non–MT-mediated cellular protection mechanisms. In addition, a revertant cell line (CdR-rev) was developed and proved to be a powerful tool for the determination of gene expression changes relevant or incidental to development of the Cd2+ resistance phenotype. Through functional and molecular analysis of the differences between the revertant, resistant, and parental MT(–/–) cell lines, we have significantly extended previous work (Yanagiya et al., 1999, 2000) by more precisely defining the transport process involved in cellular Cd2+ uptake. Genetic, biochemical, and functional analyses suggest that the T-type Ca2+ channel, Cacnα1G, is an important pathway for cellular entry of Cd2+. Likewise, evidence implies that decreased expression of Cacnα1G is a protective mechanism for cells exposed to Cd2+. Decreased Cacnα1G expression may be a key factor in the cellular response to Cd2+ exposure and may provide for acquired tolerance in cells that poorly produce MT. Given the frequently observed interindividual variability of MT expression seen in human subjects (Onosaka et al., 1985; Bem et al., 1988; Silevis-Smitt et al., 1992; Yoshida et al., 1998; Allan et al., 2000; Wu et al., 2000), knowledge of secondary factors in acquired Cd2+ tolerance could be very important.
Previous studies have suggested that Cd2+ can enter cells through L-type Ca2+ channels (Friedman and Gesek, 1994; Souza et al., 1997), and in the current study, uptake of Cd2+ was inhibited by verapamil, an L-type Ca2+ channel antagonist. However, verapamil has also been reported to inhibit T-type Ca2+ channels at concentrations as low as 10 μM, whereas more specific inhibition of L-type channels occurs at low micromolar concentrations (Heady et al., 2001). Consistent with the possibility of non–L-type channel inhibition by verapamil was the observation that two other L-type channel antagonists, diltiazem and nicardipine, had no effect on Cd2+ uptake. In contrast with verapamil, mibefradil blocks T-type Ca2+ channels 10 to 30 times more potently than L-type Ca2+ channels (Heady et al., 2001). Electrophysiological experiments using a variety of T-type Ca2+ currents have shown mibefradil IC50 values ranging from 0.1 to 4.7 μM (Heady et al., 2001). Thus, the ability of mibefradil to inhibit Cd2+ uptake by the MT(–/–) parental cell line with an IC50 of 5 μM is consistent with inhibition of a T-type Ca2+ channel. Unlike other members of the T-type Ca2+ channel protein family, Cacnα1G, has been shown to be relatively insensitive to Ni2+ inhibition (Lacinová et al., 2000). Thus, it has been previously reported that Ni2+ inhibits Cacnα1G with an IC50 value of 470 μM, comparable with the inhibitory potency of Ni2+ observed in the MT(–/–) parental cell line (IC50 = 300 μM) (Lacinová et al., 2000). Combined with these previous results, the present data implicate T-type Ca2+ channels as a critical component of Cd2+ entry into cells.
Uptake of Cd2+ was not affected by the metabolic inhibitors rotenone or sodium azide, suggesting a passive mechanism, consistent with a channel. T-type Ca2+ channels are “activated” or opened by low-voltage currents close to the resting membrane potential of the cell. High extracellular concentrations of K+ are often used as a depolarizing stimulus to activate voltage-dependent Ca2+ channels and therefore increase Ca2+ influx into cells (Jagannathan et al., 2002). Thus, our observation that Cd2+ uptake by MT(–/–) parental cells was unchanged by increased K+ concentrations is inconsistent with a voltage activation mechanism. However, previous studies have shown that recombinant Cacnα1G channels expressed in human embryonic kidney 293 cells are insensitive to increased K+ concentrations, unless cell membranes contain endogenous ion channels, such as Kir2.1, that stabilize resting membrane potential (Kim et al., 2004). Therefore, the uptake of Cd2+ in our cell system is possibly not affected by increased K+ concentrations, because such endogenous ion channels are absent. Indeed, preliminary real-time RT-PCR analysis of Kir2.1 expression in the MT(–/–) cell line suggested that it is present at very low levels (data not shown).
In vivo, Cacnα1G is expressed in many brain regions with especially high levels in amygdala, thalamus, subthalamic nuclei, and cerebellum (Perez-Reyes et al., 1998; Yunker et al., 2003). In addition, Cacnα1G is expressed at lower levels in heart, placenta, lung, and kidney (Perez-Reyes et al., 1998; Andreasen et al., 2000). In the rat, Cacnα1G mRNA is expressed in all regions of the kidney, including the distal and proximal tubules. Immunohistochemistry of rat kidney showed that Cacnα1G is localized to the apical plasma membrane of distal convoluted tubule, connecting tubule principal cells and inner medullary collecting duct principal cells (Andreasen et al., 2000). Cd2+-induced renal injury is dependent upon uptake and accumulation of this metal and although speculative, expression of Cacnα1G in this tissue could have implications for Cd2+ toxicity (Friedman and Gesek, 1994).
Dmt1/Slc11a2 is a transporter expressed at the apical surface of cells in many mammalian tissues and at particularly high levels in the small intestine and kidney (Gunshin et al., 1997). Evidence strongly suggests that Dmt1/Slc11a2 plays an important role in the absorption of ferrous iron and potentially other divalent metals in the proximal duodenum (Canonne-Hergaux et al., 1999; Leazer et al., 2002). Although Dmt1/Slc11a2 is a candidate transporter of Cd2+, uptake in the MT(–/–) parental cell line indicated that this process was not Dmt1/Slc11a2-mediated. For example, uptake of Cd2+ by the MT(–/–) cell line was potently inhibited by Zn2+, maximal at slightly acidic to neutral pH, insensitive to Fe2+, and mediated with very high affinity. In contrast, transport by Dmt1/Slc11a2 is insensitive to Zn2+, inhibited by Fe2+, maximal at pH 5.5, and mediated at lower affinity (Km = 1 μM) (Gunshin et al., 1997; Okubo et al., 2003). Gene expression analysis indicated that there was no difference in the Dmt1/Slc11a2 mRNA expression in MT(–/–) parental versus the CdR or CdR-rev cell lines consistent with the non-Dmt1/Slc11a2 phenotype.
Experiments conducted by Yanagiya et al. (2000) using an independently derived Cd2+-resistant MT(–/–) cell line also suggested that a non-Dmt1/Slc11a2 Cd2+ uptake mechanism was present in the parental MT(–/–) cell line. Their Cd2+-resistant cell line had a reduced ability to take up Mn2+, and Mn2+ was a potent inhibitor of Cd2+ uptake (Ki = 0.14 μM) (Yanagiya et al., 2000). Thus, the authors concluded that the MT(–/–) cell line expresses a novel high-affinity Mn2+ transport system through which Cd2+ can enter the cell. In the current study, expression of AE4/Slc39a7, a member of the SLC39 family of metal ion transporters, a proposed Mn2+ transporter (Eide, 2004), was expressed at similar levels in CdR and MT(–/–) parental cell lines, suggesting it is not involved in the observed alterations in Cd2+ uptake. Very little is known about the molecular pathways by which Mn2+ enters mammalian cells. Although further experiments are required to be conclusive, there are examples of overlap in Ca2+ and Mn2+ uptake (Van Baelen et al., 2004); Mn2+ has been shown to permeate through a related T-type Ca2+ channel, Cacnα1H (Kaku et al., 2003); and potentially a common mechanism exists (i.e., Cacnα1G) for the passage of Cd2+, Ca2+, and Mn2+ into the MT(–/–) cell line.
The aquaporins are a family of membrane proteins that allow rapid transport of water across lipid membranes (Law and Sansom, 2002). Certain aquaporins, the aquaglyceroporins, will also allow the downhill movement of uncharged solutes such as glycerol and urea (Liu et al., 2004). It is noteworthy that several aquaglyceroporins, including human AQP7 and AQP9, have been shown to be important for arsenic passage into cells (Liu et al., 2004). Analysis of CdR1, CdR2, and MT(–/–) parental cell lines indicated that the expression of aquaporin 1 was decreased in the CdR versus the parental cell line and that aquaporin 1 expression was intermediate in the CdR-rev cell line, consistent with the intermediate Cd2+ resistance phenotype. The functional implications of the decreased RNA expression of aquaporin 1 are not clear. Unlike the aquaglyceroporins, aquaporin 1 transport has been shown to be highly selective for water molecules (Law and Sansom, 2002). The high-resolution X-ray structure of bovine aquaporin 1 (Sui et al., 2001) indicates that the channel pore is not wide enough to allow the passage of a hydrated ion, such as Cd2+, and not energetically favorable for dehydration to occur before entering the channel (Law and Sansom, 2002). Therefore, it is highly unlikely that aquaporin 1 is directly involved in translocation of Cd2+ across the plasma membrane, but it could have a different function such as in osmoregulation. Indeed, aquaporin 1 is lost from the brush-border membrane of kidney proximal tubules of rats treated with Cd2+, suggesting that this channel is affected by the metal in vivo (Sabolic et al., 2002).
In conclusion, understanding non–MT-mediated cellular protection mechanisms is important because of the interindividual variability in MT expression in humans. In addition, such mechanisms could work in synergy with MT to provide protection from this toxic metal. This work clearly indicates that decreased cellular uptake of Cd2+ is a very important mechanism of acquired Cd2+ resistance. The thorough characterization of Cd2+ transport in MT(–/–) cells has lead to the identification of Cacnα1G as a potential pathway for cellular uptake of Cd2+. Although further research is required to understand the physiological relevance of our observations, down-regulation of Cacnα1G expression could be very important for cellular protection from Cd2+ under normal circumstances and for persons with MT(–/–) deficiencies.
Acknowledgments
We thank Dr. John S. Lazo (University of Pittsburgh, Pittsburgh, PA) for the kind gifts of the MT(–/–) and MT(+/+) cell lines. We thank Dr. Maureen W. McEnery (Case Western Reserve University, Cleveland, OH) for the generous gifts of the CW53 antibody and murine cerebellum homogenates. We also thank Drs. Larry Keefer, Wei Qiu, and Jean Francois Coppin for critical review of the manuscript.
Footnotes
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This research was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research. E.M.L. is the recipient of a Canadian Institutes of Health Research Postdoctoral Fellowship and a Davies Charitable Foundation Research Fellowship Award.
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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doi:10.1124/mol.105.014241.
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ABBREVIATIONS: MT, metallothionein; Dmt, divalent metal transporter; CdR, Cd2+-resistant; Cdr-rev, Cd2+-resistant revertant; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; RT-PCR, reverse transcriptase-polymerase chain reaction; Ct, cycle time; Zirtl, zinc-iron regulated transporter-like.
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↵1 Current affiliation: School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina.
- Received May 22, 2005.
- Accepted November 10, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
References
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