ToxSci Advance Access originally published online on June 20, 2007
Toxicological Sciences 2007 99(1):267-276; doi:10.1093/toxsci/kfm158
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Perturbation of Copper (Cu) Homeostasis and Expression of Cu-Binding Proteins in Cadmium-Resistant Lung Fibroblasts
* Department of Biochemistry
Department of Microbiology, Boston University School of Medicine, 715 Albany Street, Boston, Massachusetts 02118
1 To whom correspondence should be addressed. Fax: (617) 638-5339. E-mail: wandeli{at}bu.edu.
Received May 21, 2007; accepted June 8, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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To probe mechanisms of cadmium (Cd) damage to the lung extracellular matrix (ECM), we developed Cd-resistant (CdR) rat lung fibroblasts (RFL6) by incubation with graded concentrations of Cd. CdR cells downregulated lysyl oxidase (LO), a copper (Cu)-dependent enzyme essential for crosslinking of collagen and elastin in the ECM, in conjunction with upregulation of other Cu-binding proteins including Cu,Zn-superoxide dismutase (SOD1), copper chaperone for SOD1 (CCS1), metallothionein (MT), and Menkes P-type ATPase (ATP7A), a Cu transporter in the membrane of the Golgi apparatus, as well as
-glutamylcysteine synthetase (-GCS), an enzyme for glutathione biosynthesis. Reduction and loss of cytoplasmic distribution of LO in CdR cells were accompanied by its dislocation with the Menkes P-type ATPase and the endoplasmic reticulum marker. CdR cells displayed a defect in LO catalytic activity but an enhancement in Cu,Zn-SOD catalytic activity consistent with the protein expression levels of these enzymes. Although long-term Cd exposure of cells enhanced the Menkes P-type ATPase protein expression, actually, it reduced Cu-dependent catalytic activity of this enzyme in parallel with the deficiency of LO. The low level of 64Cu bound to the LO fraction and the high level of 64Cu bound to the MT fraction provide direct evidence for limitation of Cu bioavailability for LO existing in the CdR cells. These results suggest that downregulation of LO is linked with upregulation of other Cu-binding proteins and with alteration in Cu homeostasis in the CdR phenotype. Key Words: lysyl oxidase; cadmium; copper; metallothionein; Menkes P-type ATPase; copper-binding proteins.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Lysyl oxidase (LO), a copper (Cu)-dependent enzyme, catalyzes crosslinking of collagen and elastin critical for extracellular matrix (ECM) morphogenesis and tissue repair in the lung and other organs. LO is synthesized by fibroblasts as a 46-kDa preproenzyme. Following signal peptide cleavage and N-glycosylation, the resulting 50 kDa proenzyme is secreted and then proteolytically cleaved to the 32-kDa functional species in the ECM (Li et al., 1995
). LO specifically binds 1 mol of Cu(II) at its active site per mole of enzyme. Cu binding to the proenzyme of LO occurs in secretory vesicles such as the trans-Golgi apparatus (Kagan and Li, 2003). The activity of purified LO can be totally inhibited by metal chelators, e.g., 
, '-dipyridyl (Gacheru et al., 1990). Dietary deprivation of Cu induced lathyritic injuries in the lung of animal models due to suppression of LO activity (Dubic et al., 1985; Harris, 1986).
Cu, an essential trace element, acts as a cofactor for approximately 30 enzymes such as LO (Harris, 1991). To maintain physiological functions and avoid its toxic effects, generally, Cu is sequestered in nonreactive forms as it is transported into cells and through cellular compartments (Valentine and Gralla, 1997). For example, copper chaperone for Cu,Zn-superoxide dismutase (CCS1) delivers Cu to Cu,Zn-superoxide dismutase (SOD1) in the cytosol (Culotta et al., 1997); COX17 directs Cu to the mitochondria for activation of cytochrome oxidase (CCO) (Punter et al., 2000); and Hah1 specifically carries Cu to the secretory pathway for incorporation into Cu enzymes destined for the cell surface or extracellular space (Klomp et al., 1997). The target of Cu delivery by Hah1 is an intracellular transmembrane P-type ATPase encoded by the Menkes and Wilson disease genes (Packman et al., 1994; Cox and Thomas, 1994). The lack of the P-type ATPase in the membrane of secretory vesicles such as trans-Golgi apparatus induces Cu transport deficiency leading to abnormal Cu accumulation in Menkes cells, limitation of Cu bioavailability for secretory Cu enzymes, e.g., LO, and eventual inactivation of these enzymes (Packman et al., 1994).
Cadmium (Cd) is a toxic metal but still widely used in industries. Occupational exposure to Cd occurs mainly in the form of airborne dusts and fumes affecting an estimated 510,000 workers in the United States (IARC, 1993). In addition to the occupational exposure, cigarette smoke constitutes a major source of Cd exposure for humans since tobacco leaves naturally accumulate Cd. The lung, a major Cd-target organ, not only absorbs but also accumulates Cd with a biological half-life of 9.4 years (IARC, 1993). Cd exposure induces various pathological lesions in the lung such as emphysema (Lane and Campbell, 1954). Our previous studies investigating the phenotypic changes in rat fetal lung fibroblasts (RFL6) following long-term exposure to graded concentrations of Cd have shown that Cd-resistant (CdR) cells exhibited downregulation of LO coupled with upregulation of metallothionein (MT) and glutathione (GSH), two Cu-scavenging agents (Zhao et al., 2006). To further probe molecular mechanisms of Cd damage to lung LO, we have compared expression of LO with other Cu-binding proteins and examined Cu cellular uptake and distribution in CdR cells, a chronic Cd exposure model.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Materials.
Cadmium chloride, 99.9% pure, was from Aldrich Chemicals (Milwaukee, WI). A rabbit anti-LO antibody was prepared and employed as a probe of LO protein as previously described (Li et al., 1995
). A mouse anti-MT antibody was from Dako Inc. (Fort Collins, CO). A rabbit anti-Cu,Zn-SOD antibody and a rabbit anti-CCS1 were from Santa Cruz Biotech. (Santa Cruz, CA). A rabbit anti-Mn-SOD antibody was from Stressgen Biotechnologies (Victoria, British Columbia, Canada). A chicken anti-Menkes P-type ATPase-ATP7A antibody was from Chemicon International (Cambridge, MA). A rabbit anti--glutamylcysteine synthetase (-GCS) was from NeoMarkers (Fremont, CA). The endoplasmic reticulum (ER) marker [DiOC6(3)] was from Molecular Probes Inc (Eugene, OR). Horseradish peroxidase (HRP)–conjugated goat anti-rabbit, -mouse, or -chicken IgG was from Santa Cruz Biotech. Cy3-conjugated goat anti-rabbit IgG and Cy2-conjugated Goat anti-chicken IgG were from Jackson Immuno Research Laboratories, Inc. (West Rove, PA). 64Cu was obtained from Edward Mallinckrodt Institute of Radiology, Washington University School of Medicine (St Louis, MO). CNBr-activated Sepharose 4 fast flow beads were from Pharmacia Biotech (Uppsala, Sweden). Protein assay reagent was from Pierce (Rockford, IL). All tissue culture products were from GIBCO (Grand Island, NY).
Cell culture and treatment.
RFL6 cell line derived from the normal Sprague-Dawley rats was obtained from American Type Culture Collection and maintained in 10% fetal bovine serum (FBS) in Dulbecco's modified Eagle's medium (DMEM). Cadmium resistance of cells was achieved by serial passages of cells exposed to graded concentrations of CdCl2 from 10 to 80µM as described (Li et al., 1995). Cells with different degrees of Cd resistance were referred to as CdR10, CdR20, CdR30, CdR40, CdR50, CdR60, and CdR80 cells representing these cells resistant to 10, 20, 30, 40, 50, 60, and 80µM Cd, respectively. Cd-sensitive (CdS) RFL6 and CdR cells were seeded at 8 x 104 in 100-mm dish and grown in 10% FBS for 1 day. Cells were then growth-arrested by incubating in 0.3% FBS/DMEM for 3 days followed by changing to a fresh 0.3% FBS/DMEM without or with 5µM CdCl2 for CdS RFL6 cells or different concentrations of CdCl2 for CdR cells and incubating for 24 h.
Western blot analysis.
Cells were washed twice with PBS, scraped, and stored in aliquots at – 20°C. Thawed cells were lysed in the RadioImmuno Precipitation Assay buffer composed of 1x PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and 2M urea, pH 7.4, with protease inhibitor cocktail freshly added (Chen et al., 2005). Protein concentration in each sample was determined by the bicinchoninic acid protein assay reagents. Cell lysates containing equal amounts of protein (10–20 µg) were boiled in an SDS sample buffer and analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) gradient gel (4–15%). The separated proteins were transferred from the gel to a nitrocellulose membrane (Schleicher & Schuell, Keene, NH). Nonspecific binding sites were blocked by incubating the nitrocellulose membrane in Tris-buffered saline containing 0.1% Tween 20 with 5% nonfat dry milk. Membranes were then incubated overnight at 4°C with primary antibodies as follows: a rabbit anti-LO (1:1000), a mouse anti-MT (1:500), a rabbit anti-CCS1 (1:1000), a rabbit anti-Cu,Zn-SOD (1:1000), a rabbit anti-Mn-SOD (1:1000), a rabbit anti--GCS (1:500), and a chicken anti-ATP7A (1:500). They were rinsed with Tris-buffered saline with 0.1% Tween 20 three times each for 5 min and then incubated with HRP-conjugated corresponding secondary antibodies, i.e., anti-rabbit, anti-mouse, and anti-chicken IgGs with 1:10,000, 1:5000, and 1:10,000 dilutions, respectively, for 1 h at room temperature. After washing, blots were developed with an enhanced chemiluminescence system (PerkinElmer Life Sciences, Boston, MA). Molecular weights were determined by comparison with BenchMark prestained protein ladder (Invitrogen, Carlsbad, CA). Protein bands were quantitated by the 1 D Scan EX software (Scananalytics, Fairfax, VA). Experiments as shown here and below were repeated at least three times with reproducible results, and a representative one is presented, unless otherwise indicated.
Immunocytochemistry and fluorescence microscopy.
Growth-arrested control, Cd treated, and CdR cells on coverslips were washed twice with PBS and fixed with cold (– 20°C) methanol for 5 min. To prevent nonspecific antibody binding, cells were blocked with a 5% normal goat serum for 10 min. Cells were double stained sequentially first with a rabbit anti-LO (1:100) antibody followed by an anti-rabbit IgG-Cy3–conjugated (1:500) secondary antibody. They were then stained either with a chicken anti-ATP7A (1:200) antibody followed by an anti-chicken IgG-Cy2–conjugated (1:200) secondary antibody or with the ER marker, i.e., DiOC6(3), fluorescent dye, at a final concentration of 0.5 µg/ml in PBS for 5 min at room temperature. Cell-associated fluorescent signals were examined under Nikon TE-2000 microscope.
LO activity assay.
Growth-arrested CdS and CdR cells were incubated in 0.3% FBS/phenol red–free DMEM for 24 h. The conditioned medium was collected and assayed for LO activity using diaminopentane as a substrate and Amplex red as a hydrogen peroxide probe as described (Palamakumbura and Trackman, 2002). In a typical assay, samples (e.g., 500 µl conditioned medium) were mixed with the reaction mixture containing 0.05M sodium borate, pH 8.2, 10mM diaminopentane, 10µM Amplex red, 40 µg HRP, and 1.2M urea in a final volume 2 ml in the presence or absence of 0.5mM ß-aminopropionitrile (BAPN), an active site inhibitor of LO. H2O2 release was continuously monitored for at least 300 s at excitation and emission wavelengths of 563 and 587 nm, respectively, at a constant temperature of 37°C, as specified in the thermostatted cuvette chamber of an LS 55 Luminescence Spectrometer (PrekinElmer Instruments, Shelton, CT). All enzyme activities were expressed as fluorescence values at 300 s after the reaction, corrected for background levels of H2O2 release determined in the reaction mixture supplemented with BAPN, and normalized to total cell protein.
SOD activity assay.
Growth-arrested CdS and CdR cells were homogenized in 0.5% NP40/PBS, and cytosolic fractions were isolated following microcentrifugation. The SOD activity in the cell extract was determined by measuring its inhibition of auto-oxidation of epinephrine to produce adrenochrome as described (Misra and Fridovich, 1972). In the typical assay, samples 100 µl each were mixed with the reaction mixture containing 3x 10–4M epinephrine, 1x 10–4M EDTA, and 0.05M sodium carbonate, pH 10.2, in a final volume of 1 ml. The production of adrenochrome upon the reaction was monitored at least 5 min at A480 nm at 30°C using Beckman Coulter DU-530 spectrophotometer (Beckman Instruments, Fullerton, CA). A standard curve using bovine SOD (Sigma, St Louis, MO) was always generated with assays, and results were expressed as units of SOD/mg total cell protein.
Cu-ATPase activity assay.
Golgi apparatus–enriched fractions were isolated from growth-arrested CdS and CdR cells as described (Warley and Cook, 1976). Briefly, cells were homogenized in 1 ml 0.5M sucrose containing 37.5mM Trizma, pH 6.4, 1% dextran, and 5mM MgCl2. Homogenates (1 ml) were layered directly on a discontinuous gradient consisting of 1.1M sucrose (2 ml) layered over 1.25M sucrose (2 ml), both solutions containing 37.5mM Trizma, pH 6.4, 1% dextran, and 5mM MgCl2, followed by centrifugation at 99,000x g at 4°C for 5 h. Golgi apparatus–enriched materials collected at the 0.5M/1.1M sucrose interface were treated with penicillamine (3.75 µmol/mg protein) and mercaptoethanol (1.46 µg/mg protein), then mixed with water, and pelleted by centrifugation for 30 min at 99,000x g. Pellets were resuspended in a 50mM Tris buffer, pH 7.2, containing 250mM sucrose and 10mM Hepes. In a typical Cu-ATPase assay (Usta et al., 1997), the reaction mixture contained 7.5mM Trizma, pH 7.2, 2mM ouabain, 1.4 µg oligomycin, 3mM MgCl2, 10 µg Golgi apparatus protein in the presence or absence of 300µM CuCl2 in a final volume of 0.3 ml. The reaction was started by the addition of ATP to a final concentration of 3mM, carried out at 37°C for 10 min and terminated by addition of trichloroacetic acid. The amount of inorganic phosphate (Pi) released was measured colorimetrically using the malachite green assay as described (Lanzetta et al., 1979). The Pi assay reagent was prepared by mixing three parts of 0.045% malachite green hydrochloride with one part of 4.2% ammonium molybdate in 4M HCl and 0.06% Tween 20 followed by filtration. One hundred and fifty microliters of the reaction mixture were mixed with 540 µl of Pi reagent solution. After 1 h incubation, the color was read at A660 nm in Beckman Coulter DU-530 spectrophotometer. The activity of Cu-ATPase was expressed as µM Pi/mg total cell protein above the basal Mg-ATPase activity using phosphate as standard.
Assay for cellular uptake and LO or MT binding of Cu.
For cellular Cu uptake assay (Schmitt et al., 1983), growth-arrested RFL6 and CdR40 cells were pulse labeled with 64Cu (40 µCi/ml) in phosphate buffered saline containing 10mM glucose (PBSG) for 1 h, trypsinized, and centrifuged. Cell pellets were then lysed in 0.2% Triton X-100/PBS and aliquots of lysates counted for -radioactivity. To assay for LO or MT binding of Cu, both RFL6 and CdR40 cells were incubated with the same amount of 64Cu (40 µCi/ml) in PBSG for 4 h. Cells were harvested by scraping and lysed in 10mM HEPES (pH 7.4)/50mM NaCl/1mM PMSF/0.2% Triton X-100/2M urea by sonication. For isolation of LO fraction, homogenates with the same radioactivities were mixed with LO antibody–coupled Sepharose beads (Pharmacia Biotech., Uppsala, Sweden) prepared as described (Harlow and Lane, 1988) with constant agitation for 2 h at room temperature. Beads were sequentially washed with lysis buffer, lysis buffer omitting Triton X-100, and finally with 4M urea/16mM potassium phosphate (pH 7.8) for 1 h with shaking. The urea-released protein fractions were measured for radioactivities by -counting and for protein levels by UV280. After isotopic decay, same volume of urea-released proteins from RFL6 and CdR cells were analyzed on 10% SDS-PAGE followed by Western blot. For isolation of the MT fraction, homogenates from both CdS and CdR cells were mixed with MT-antibody–coupled Sepharose beads, washed and collected as described above for isolating LO fractions.
Statistical analysis.
Data were expressed as mean ± SD of at least three independent experiments. Statistical differences between means were determined using one-way ANOVA followed by Bonferroni's post hoc test or two-tailed Student's t-test when appropriate. A p value < 0.05 was considered significant.
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RESULTS |
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Comparison of the Expression of LO to Other Cu-Binding Proteins as well as
-GCS in CdS and CdR CellsLO is a Cu-dependent enzyme. Downregulation of LO was shown as a major phenotype change in CdR cells following long-term Cd exposure. To answer the question whether other Cu-binding proteins were also perturbed in the course of Cd resistance, we compared by Western blot the expression of LO with Cu,Zn-SOD, CCS1, Menkes P-type ATPase, MT as well as
-GCS, an enzyme essential for synthesis of the metal-binding tripeptide GSH in CdR cells with different degrees of Cd resistance. As shown in Figure 1A, LO antibody immunoreactive proteins in RFL6 cells include a 46-, a 50-, and a 32-kDa band in the cell extract representing a typical protein profile of LO synthesis and processing by fibrogenic cells, i.e., RFL6 lung fibroblasts initially synthesize a 46-kDa preproenzyme; after signal peptide cleavage and N-glycosylation, the resulting 50 kDa proenzyme is secreted and then proteolyzed to the 32-kDa functional species extracellularly (Li et al., 1995; Zhao et al., 2006). Since a part of the mature enzyme was attached to the cell membrane and the ECM, the 32-kDa protein was positively detected in the cell extract fraction. Comparatively, CdR cells displayed markedly decreased levels in the 46-, the 50-, and the 32-kDa proteins associated with appearance of a conspicuous 52 kDa protein that was recognized by the anti-LO antibody. The densitometry analysis showed that the 46-kDa preroenzyme, the 50-kDa proenzyme, and the 32-kDa mature enzyme were inhibited in different degrees in the same CdR cell type, and the 32-kDa species is essentially undetectable in CdR50, CdR60, and CdR80 cells. These results suggest that CdR cells exhibited inhibition of LO synthesis and processing following long-term Cd exposure. The 52-kDa protein may represent an abnormal processing product of LO. In sharp contrast, there were significant increases at the top levels of other Cu-binding proteins observed in the CdR phenotype as exemplified by Cu,Zn-SOD, an enzyme catalyzing the destruction of O2– free radicals, reaching 2.2-fold (Fig. 1B); Mn-SOD reaching 1.54-fold (Fig. 1B); CCS1, a copper chaperone for Cu,Zn-SOD, reaching 1.8-fold (Fig.1C); Menkes P-type ATPase, i.e., ATP7A, a transmembrane enzyme carrying Cu to the secretory pathway, reaching 8.5-fold (Fig. 1D); and -GCS, a rate-limiting enzyme for GSH synthesis, reaching 1.62-fold (Fig. 1E), of corresponding RFL6 controls, respectively. Furthermore, MT, a metal-binding protein essential for cellular Cu storage and transport, was elevated from a nondetectable level in RFL6 cells to an extremely high level in CdR cells in a dose-dependent manner (Fig. 1F). For example, the cellular MT was amplified by 126-fold in cells resistant to Cd from 10 to 80µM. These results indicate that not all Cu-binding enzymes or proteins were suppressed in the CdR cells. Thus, downregulation of LO may be a specific phenotype change for cell resistance to Cd.
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Alterations in LO Colocalization With the Menkes P-Type ATPase and the ER Marker in CdR Cells
Secreted proteins are synthesized in the ER. They commonly undergo posttranslational modifications in the secretory pathways from the ER, Golgi apparatus, exocytotic vesicles to the cell membrane. To characterize alterations of LO localization in the CdR phenotype, we visualized LO and Menkes P-type ATPase, a Cu-transporter in the Golgi apparatus membrane (Dierick et al., 1997
), or the ER (Linder and Goode, 1991) in the same cell by fluorescently double staining. Figure 2A shows the well-spread RFL6 cells containing numerous, red LO-positive fragments and spots which were generally concentrated in the central area of the cell and extended to the cell periphery, while the green color Menkes P-type ATPase (ATP7A) signals were distributed mainly in the cytoplasm with a high density in the perinuclear area. Superimposition of images showed extensive coincidence of red and green color signals forming orange or yellow patches, fragments, and spots. These results indicate that LO-positive fragments or spots were mostly colocalized with the Menkes P-type ATPase. RFL6 cells treated with 5µM Cd for 24 h often lack the typical, short LO-positive fragments and spots, instead, few irregular patches of LO-positive staining appeared in the nuclei or asymmetrically distributed in the cytoplasm. Note that under this condition, some irregular LO patches still overlapped with the Menkes P-type ATPase staining. In contrast, CdR40 and CdR80 cells following long-term Cd exposure displayed a distinctly different localization of LO proteins. Significantly decreased red LO signals were mostly scattered within the nuclei, while the green P-type ATPase signals were aggregated to form a ring or half ring patch located on the perinuclear area. There was little coincidence of LO with Menkes P-type ATPase in the CdR phenotype as evidenced by near absence in CdR80 cells of the orange or yellow color spots or patches, a signal for colocalization of LO with Menkes P-type ATPase. Furthermore, the codistribution and the separative localization of LO (red signal) with the ER marker (green signal) were also observed, respectively, in CdS and CdR phenotypes (Fig. 2B). Thus, markedly reduced LO signals in the cytoplasm concomitant with the localization of remaining enzyme mainly within the nuclei are an important morphological alteration of CdR cells in comparison to parental CdS control.
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Comparison of Catalytic Activities of LO to Menkes P-Type ATPase and Cu,Zn-SOD
To assess changes in functionality of LO, Menkes P-type ATPase, and Cu,Zn-SOD in the course of cell CdR conversion, we further examined catalytic activities of these enzymes in CdS and CdR cells. Since LO activity was mainly present in the conditioned media, Menkes P-type ATPase activity in the Golgi apparatus, and Cu,Zn-SOD activity in the cytoplasm, those cell fractions were isolated and tested. As shown in Figure 3, LO catalytic activities in CdR cells were much less than that in CdS cells from 41% of the control in CdR10 cells (p < 0.05) to the maximal inhibition amounting to 5% of the control in CdR80 cells (p < 0.001). In contrast, catalytic activities of SOD1, a Cu-dependent antioxidant enzyme, were significantly elevated in cells following the development of Cd resistance. For example, CdR20, CdR40, and CdR80 cells increased Cu,Zn-SOD activities to 152% (p < 0.05), 179% (p < 0.01), and 210% (p < 0.001) of the control, respectively. Notably, CdR cells expressed high levels of the Menkes P-type ATPase protein but low levels of this enzyme activity in comparison to the CdS controls. A 50% decrease in Cu-dependent P-type ATPase activity was observed in CdR20 cells. Stronger Cd resistance of cells was accompanied by more serious inhibition of enzyme activity such that the Cu-dependent P-type ATPase activity was suppressed by 85% in CdR80 cells. These results indicate that LO and Cu-dependent P-type ATPase activities were inhibited, while Cu,Zn-SOD activity was enhanced; thus, not all Cu-dependent enzymes were suppressed at the catalytic level in the CdR phenotype.
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64Cu Uptake and Binding to LO and MT in CdS and CdR Cells
To directly assess the abnormal Cu metabolism in CdR cells, we examined 64Cu uptake and binding to LO and MT fractions in CdS and CdR cells. Because of the short half-life of 64Cu (12.5 h) and limiting amount of isotope available, these experiments were done only in CdS and CdR40 cells. As shown (Fig. 4A), after 1 h pulse, the amount of 64Cu radioactivity associated with CdR40 cells was 213% of the CdS control. Thus, the presumed limitation of Cu bioavailability for LO in these cells is not due to a reduced Cu uptake. The high level of Cu ions accumulated in CdR cells may result from the elevated levels of cellular thiols, e.g., MT and GSH, which bind Cu with high affinity (Kagi and Schaffer, 1988
; Harris, 1991). Using immunoprecipitation by antibody-coated beads and urea elution procedure, the Cu-bound LO fraction was successfully isolated from the 4 h 64Cu–pulsed cells and conditioned media. In contrast to Cu uptake, an extremely low level of 64Cu bound to the LO fraction was observed in CdR cells, amounting to only 9% of the CdS control. Thus, a severe limitation of Cu bioavailability for LO was characterized in this CdR phenotype. Moreover, the eluted protein profile (see the inserted figure in Fig. 4A) showed that a high density of the 52-kDa protein band appeared in the CdR cell extract with the low densities of the 50- and 32-kDa species which were predominant in CdS cells, further indicating the LO deficiency at the protein level in CdR cells as presented in Figure 1A. Furthermore, using the identical immunoprecipitation method, the Cu-bound MT fraction was isolated from the 4 h 64Cu–pulsed CdS and CdR40 cells. As shown in Figure 4B, an extremely high level of 64Cu was associated with the MT fraction in CdR cells amounting to 14-fold of the CdS control. These results strongly support the conclusion that LO has a relatively low affinity for Cu in comparison to MT since they were simultaneously present in the CdR cell extract.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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LO is a Cu-dependent enzyme critical for ECM morphogenesis and repair by crosslinking of collage and elastin. In this study, we demonstrate that the phenotype change of rat lung fibroblasts from CdS to CdR following long-term Cd exposure was manifested by downregulation of LO protein in conjunction with upregulation of various Cu-binding proteins including Cu,Zn-SOD, CCS1, MT, and Menkes P-type ATPase as well as
-GCS. Loss of cytoplasmic distribution of LO in CdR cells was accompanied by loss of its colocalization with the Menkes P-type ATPase and the ER marker. The CdR cells displayed a decreased LO enzyme activity but an increased Cu,Zn-SOD enzyme activity consistent with alterations in protein levels of these enzymes. Although long-term Cd exposure enhanced the Menkes P-type ATPase protein expression, actually, it damaged functions of this enzyme parallel with the defect in LO catalytic activity in the CdR phenotype. The low level of Cu bound to the LO fraction and the high level of Cu bound to the MT fraction provide direct evidence for the abnormal Cu metabolism existing in the CdR cells. Thus, a severe limitation of Cu bioavailability for LO is present in the CdR phenotype.
Cu acts as a cofactor essential for catalytic activities of many cellular enzymes such as LO, Menkes P-type ATPase, and SOD1. Major cellular Cu trafficking pathways and LO synthesis and processing are summarized in Figure 5. Cu uptake, mediated by the cell membrane Cu transporter (Ctr), is eventually deployed to cytosolic Cu,Zn-SOD via the CCS1, to the secretory pathway for exocytosis or for incorporation into secretory Cu enzymes, e.g., LO, via the Hah1 and P-type ATPases, e.g., ATP7A, and to mitochondrial CCO via the COX17. Cytosolic concentrations of free Cu are maintained at very low levels (< 10–18M) (O'Halloran and Culotta, 2000) by Cu transport systems and metal scavenging systems such as MT and GSH. Cu effects on LO expression are proposed at the transcriptional level by its stimulation of the metal transcriptional activator which reacts with the metal regulatory element in the promoter region of the LO gene. Cu binding to LO is proposed to occur at the ER or the Golgi apparatus or both. The key components of cells that require Cu as shown in Figure 5 such as LO, P-type ATPase, Cu,Zn-SOD, CCS1, and MT as well as -GCS were tested in this study.
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Cd is a toxic and carcinogenic metal. The lung is a major Cd-target organ. To probe the molecular mechanism of Cd injury to the lung ECM, we have isolated CdR cells from rat lung fibroblasts following exposure to graded concentration of Cd. Using CdR cells as a long-term Cd exposure model, we demonstrated that inhibition of LO, a Cu-dependent enzyme, is a critical phenotype change for Cd resistance (Zhao et al., 2006
). Results in this study confirm our previous findings and further reveal downregulation of LO concomitant with upregulation of other major Cu-binding proteins in CdR cells. By comparing changes in various species of LO and their cellular distributions in CdR cells with different degrees of Cd resistance, we found that the unique 52-kDa variant LO appearing in CdR cells was mainly localized within the nuclei (Fig. 1A and Figs. 2A and 2B).
Menkes P-type ATPase (ATP7A) called the Menkes protein is a transmembrane Cu transporter located in the membrane of the Golgi compartment of the secretory pathway (Dierick et al., 1997). ATP7A contains six heavy metal–binding cysteine clusters, eight transmembrane regions, and an ATPase domain (Harris et al., 1998). Like other metal transporting ATPases, the Menkes protein uses energy from ATP hydrolysis pumping Cu ions into the Golgi lumen for insertion of Cu into Cu enzymes, e.g., LO, for extracellular secretion (Vulpe and Packman, 1995). Transient phosphorylation by ATP of the Menkes protein, an essential step in the catalytic cycle of P-type ATPases, occurs in a Cu-specific and Cu-dependent manner (Voskoboinik et al., 2001). The lack of the Menkes P-type ATPase in the membrane of secretory vesicles such as trans-Golgi apparatus induces Cu transport deficiency leading to abnormal Cu accumulation in Menkes cells, limitation of Cu bioavailability for Cu enzymes, and eventually abolishing these enzyme activities (Packman et al., 1994). Thus, Cu-dependent P-type ATPase may be implicated in Cu binding to proLO in the secretory pathway. In the case of CdR cells, decreased levels of Cu-dependent P-type ATPase activity by Cd as shown in Figure 3 may be an important upstream mechanism contributing to deficiency in LO catalytic function. It should be noted that the Menkes P-type ATPase protein level was actually enhanced in the CdR phenotype. These results suggest that Cd targets the Menkes protein only at the catalytic level but not at mRNA and protein synthesis levels. Decreased Cu-dependent P-type ATPase activity by Cd may trigger a feedback regulatory mechanism enhancing Menkes protein synthesis.
SODs are metalloenzymes catalyzing the dismutation of the superoxide ion into oxygen and hydrogen peroxide. Three distinct isoforms of SOD have been identified, i.e., Cu,Zn-SOD in the cytosol, Mn-SOD within the mitochondria, and extracellular SOD also with Cu and Zn at its active site but located in the ECM (Andreadis et al., 2003). Cu,Zn-SOD is a stable homodimer held together primarily with hydrophobic contacts. Each monomer of Cu,Zn-SOD contains two metal ions, one Cu and one Zn. The catalytic Cu is bound by four histidines, i.e., His 46, 48, 63, and 120, in a distorted tetrahedral-binding geometry in the oxidized (CuII) form and in distorted trigonal planar geometry, bound by His 46, 48, and 120 in the reduced (CuI) form. Redox-active Cu participates in oxidization and reduction of superoxide by catalyzing the spontaneous transfer of an electron to the metal and to the superoxide (Rakhit and Chakrabartty, 2006). Thus, Cu plays a critical role in structure and catalytic activity of SOD. CCS1 delivers Cu to SOD in the cytosol (Culotta et al., 1997). Cu transporter proteins contain the conserved MTCXXC (X = any residue) sequence, which appears in diverse species from bacteria to humans, representing the motif of Cu-binding sites (Cox and Thomas, 1994). Cd exposure induces production of reactive oxygen species (Koizumi et al., 1996). Elevated levels of SOD and its Cu transporter CCS1 as shown in Figs. 1B and 1C and Fig. 3 provide a defense line protecting CdR cells from toxicity of high concentrations of Cd.
MT is a thiol-rich metalloprotein involved in many cellular functions including Cu transport and storage (Kagi and Schaffer, 1988). Although four MT isoforms exist in mammals (Harris, 1991), only MT-I and MT-II are expressed in the lung (Hart et al., 1996). Cd is an effective inducer of MT. MT consists of 61–62 amino acids of which 20 are cysteines (Cys). These Cys-SH groups provide metal-binding sites. The chemical structure of MT is partitioned into two separate metal-thiolate clusters: cluster A contains 11 Cys able to bind 4 mol of Cd, while cluster B contains 9 Cys able to bind 3 mol of Cd. The affinities of metal ions for the MT-binding sites are ordered as follows: Zn(II) < Pb(II) < Cd(II) < Cu(I), Ag(I), Hg(II), Bi(III) (Kagi and Schaffer, 1988). Long-term Cd exposure activated MT genes, resulting in elevated levels of MT in CdR cells (Fig. 1F) in favor of metal storage and detoxification. The higher affinity of Cu than Cd for MT is supported by our 64Cu-binding assay in which an extremely high level of radioactivity was associated with the MT fraction in CdR cells (Fig. 4B). In addition, Cd has also been reported to selectively displace Zn but not Cu in native calf liver MT which binds four molecules of Zn and three molecules of Cu (Briggs and Armitage, 1982). Because of its high affinity for Cu, increased MT levels can change cellular Cu distribution as evidenced by the addition of exogenous MT, leading to a shift of labeled 64Cu from Cu-binding protein fractions to the MT-containing fraction in cell extract (Farrell et al., 1993). MT can bind 11 or 12 Cu molecules/mol of protein (Richards, 1989). Furthermore, our study also indicates upregulation of -GCS, a key enzyme for GSH biosynthesis, in CdR cells consistent with elevated levels of GSH in the CdR phenotype as reported by us previously (Zhao et al., 2006). GSH is a thiol-containing tripeptide which plays a critical role in cellular metabolism, essential for metal transport, and heavy metal detoxification (Meister, 1984). In addition to MT, GSH also functions in cellular Cu metabolism (Harris, 1991). Since Cu preferentially binds to GSH mainly via its -SH group before engaging MT or other cellular enzymes, reduction of Cu(II) to Cu(I) by GSH is thought to be a preliminary step in cellular Cu transport (Christie and Costa, 1984). GSH can form a complex with Cu-MT, providing the biochemical basis for Cu transfer between these two cellular thiols (Brouwer et al., 1993). Depletion of cellular GSH by buthionine sulfoximine inhibited MT binding of cellular Cu ions (Freedman et al., 1989), reflecting the significance of GSH in Cu metabolism. Thus, Cd elevation of cellular thiols such MT and GSH may change cellular Cu homeostasis and thus limit Cu bioavailability for LO which uses Cu as a cofactor. This hypothesis is supported by the finding that the extremely low level of Cu radioactivity was associated with the LO fraction in CdR cells as shown (Fig. 4A). Notably, whether changes of cellular Cu homeostasis by Cd in the CdR phenotype also affect activities of other Cu-binding enzymes that were not tested in this study such as mitochondrial CCO (Fig. 5) should closely depend on their Cu affinities and corresponding Cu chaperone or transporter functions.
Our study shows that not all Cu-dependent enzymes were suppressed in CdR cells. Apparently, different Cu enzymes bind Cu with different affinities and via different cellular transport pathways (see Fig. 5). The low level of 64Cu binding to LO (Fig. 4A) strongly supports the conclusion that LO has a relatively low affinity for Cu in comparison to MT, GSH, CCS1, and Cu,Zn-SOD. Thus, a shift of Cu from LO and Menkes P-type ATPase to MT, GSH, Cu,Zn-SOD, CCS1, etc., is expected to occur intracellularly in the development course of Cd resistance. Therefore, the lack of Cu(II) availability for LO leads to the deficiency of this enzyme in CdR cells. In brief, the results present in this study illustrate that downregulation of LO is linked with upregulation of other Cu-binding proteins and with alteration in Cu homeostasis in the CdR phenotype.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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National Institutes of Health (R01-ES 11340) and the External Research Program of Philip Morris USA Inc. and Philip Morris International.
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There are no conflicts of interest.
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