Downregulation of Lysyl Oxidase and Upregulation of Cellular Thiols in Rat Fetal Lung Fibroblasts Treated with Cigarette Smoke Condensate
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《毒物学科学杂志》
Departments of Biochemistry and Microbiology, Boston University School of Medicine, Boston, Massachusetts 02118
ABSTRACT
Lysyl oxidase (LO), a copper-dependent enzyme, plays a critical role in the formation and repair of the extracellular matrix (ECM) by catalyzing the crosslinking of elastin and collagen. To better understand mechanisms of cigarette smoke (CS)-induced emphysema, we examined changes in LO and its substrates, i.e., elastin and collagen type I, the major components of cellular thiols, i.e., metallothionein (MT) and glutathione (GSH), and gamma-glutamylcysteine synthetase (-GCS), a key enzyme for GSH biosynthesis, in cigarette smoke condensate (CSC)-treated rat fetal lung fibroblasts (RFL6). Exposure of RFL6 cells to CSC decreased levels of LO catalytic activity, mRNA, and protein, i.e., the 46 kDa preproenzyme, the 50 kDa proenzyme and the 32 kDa mature enzyme in a dose-dependent manner. In addition, CSC also inhibited the expression of collagen type I and elastin, substrates of LO and important components of the lung ECM. Meanwhile, cellular thiols including MT and GSH as well as -GCS were markedly upregulated in CSC-treated cells. To evaluate modulation of LO expression by cellular thiols, we further examined the effect of increased levels of GSH on LO expression at protein and catalytic levels. Interestingly, exposure of cells to glutathione monoethyl ester, a GSH delivery system, effectively elevated cellular GSH levels and induced a dose-dependent decrease in levels of the protein species and catalytic activity of LO. These results suggest that upregulation by CSC of cellular thiols may play an important role in the downregulation of LO and subsequently destabilization of the lung ECM in CS-induced emphysema.
Key Words: cigarette smoke condensate; lysyl oxidase; glutathione; metallothionein; collagen type I; elastin.
INTRODUCTION
Lysyl oxidase (LO) (E.C. 1. 4. 3.13), a copper (Cu)-dependent amine oxidase, is initially synthesized by fibrogenic cells such as fibroblasts and vascular smooth muscle cells as a 46 kDa preproenzyme with N-terminal signal sequences which direct nascent proteins into the lumen of the rough endoplasmic reticulum. The proprotein resulting from cleavage of the N-terminal signal peptide undergoes N-linked glycosylation in the endoplasmic reticulum and the Golgi apparatus, and is then extracellularly secreted via secretory vesicles as a 50 kDa proenzyme. In the extracellular matrix (ECM), the 50 kDa proLO is further processed by proteolysis into the enzymatically active 32 kDa mature species (Trackman et al., 1992). LO catalyzes the covalent crosslinking of elastin and collagen by oxidizing peptidyl lysines in these proteins to form peptidyl -aminoadipic--semialdehyde. These aldehyde residues can spontaneously condense with neighboring peptidyl lysines or peptidyl aldehyde, leading to the formation of insoluble aggregates found in fibrillar collagen and elastin, thus stabilizing the ECM (Kagan and Li, 2003). Therefore, LO plays a critical role in lung morphogenesis and tissue repair.
There is increasing evidence that, in addition to the crosslinking of elastin and collagen in the ECM, LO also exhibits other biological functions. For example, expression of transfected, sense LO cDNA in cells transformed by p21 Ha-Ras suppresses Ha-Ras-induced tumorigenesis, indicating a Ras-suppressor effect of LO (Kenyon et al., 1991). The 32 kDa mature LO purified from bovine aorta displays strong chemotactic activities for monocytes and vascular smooth muscle cells (Lazarus et al., 1995; Li et al., 2000). LO and its oxidized substrates exist within the nuclei of cultured vascular smooth muscle cells and 3T3 fibroblasts (Li et al., 1997, 2002). Recent studies have indicated that LO oxidizes basic fibroblast growth factor and inactivates its mitogenic potential (Li et al., 2003).
Perturbation of LO expression is involved in human diseases. Increased LO activity is associated with fibrotic diseases such as lung and liver fibrosis and atherosclerosis, whereas decreased LO activity is associated with disorders of Cu metabolism like Menkes syndrome (Kagan and Li, 2003).
Long-term exposure of humans to cigarette smoke (CS) can result in emphysema. Most studies of emphysema pathogenesis have focused on the "elastase-antielastase imbalance" hypothesis (Snider, et al., 1986). Unfortunately, this hypothesis cannot explain all aspects of the emphysema-associated pathology. For example, the CS-induced emphysematous lung in guinea pigs exhibited breakdown of collagen without significant changes in elastin (Wright and Churg, 1995). Furthermore, transgenic mice that over-express the gene for human collagenase developed emphysema of early onset (D'Armiento et al., 1992). These findings suggest that the ‘elastase-antielastase imbalance’ is not the only mechanism for emphysema pathogenesis. Thus, the molecular basis for CS-induced emphysema remains unclear and requires further investigation.
Emphysema is currently defined as ‘a condition of the lung characterized by abnormal enlargement of the airspaces with destruction of the walls’ (Snider et al., 1986). The critical role of LO in emphysema development was reflected in the disruption of the lung structure in chicks, rats, and hamsters following diet-induced deficiency of Cu, a cofactor of LO (Harris, 1986; O'Dell et al., 1978; Soskel et al., 1984). These findings provide evidence that LO deficiency is associated with the pathogenesis of emphysema.
To better understand the role of LO in CS-induced emphysema, we have examined changes in LO at mRNA, protein, and catalytic levels as well as its substrates, collagen type I and elastin, and changes in key components of lung defenses against oxidants and heavy metals, i.e., cellular thiols such as glutathione (GSH) and metallothionein (MT). In the present study, we report that cigarette smoke condensate (CSC), the particulate matter of CS, inhibits the expression of LO and its substrates, collagen and elastin, but enhances the levels of cellular thiols, MT and GSH, as well as gamma-glutamylcysteine synthetase (-GCS), a key enzyme for GSH biosynthesis (Yan and Meister, 1990), in cultured rat fetal lung fibroblasts (RFL6).
MATERIALS AND METHODS
Materials. CSC was purchased from Murty Pharmaceutical Inc. (Lexington, KY). It was prepared using a Phipps-Bird 20-channel smoking machine designed for Federal Trade Commission testing. Kentucky standard cigarettes 1R3F (University of Kentucky, KY) were smoked, and the particulate matter was trapped onto filters. The amount of CSC obtained was determined by weight increase of the filter. CSC was dissolved from the filter in dimethyl sulfoxide by sonication to yield 4% solution. The average yield of CSC was 21 mg per cigarette including 1.3 mg nicotine. Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were ordered from GIBCO (Grand Island, NY). Glutathione monoethyl ester (GME) and other biochemicals were obtained from SIGMA (St. Louis, MO).
Cell culture and treatment. The fibroblastic RFL6 cell line derived from the fetal lung tissue of normal Sprague-Dawley rats was obtained from ATCC. Since RFL6 cells display the key characteristics of the pulmonary fibroblasts in expressing collagen type I, tropoelastin, and LO as shown below, they are an ideal model for this study. Cells were regularly maintained in DMEM containing 10% FBS. Cells were seeded at 8 x 104/ml in 100-mm dishes with 10 ml complete medium and incubated for 24 h. To standardize experimental conditions, cells were growth-arrested and synchronized at the G1 phase by incubation in 0.3% FBS/DMEM for 72 h, followed by changing to a fresh 0.3% FBS/DMEM and incubation for another 24 h before experiments (Li et al., 1995). Cells were treated for 24 h with CSC at final concentrations ranging from 0 to 120 μg/ml, unless otherwise indicated, then processed for further analysis. Control cells were incubated in the presence of vehicle only.
Western blot analysis. Control and treated cells were washed and lysed in the RIPA buffer composed of 1x PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 2 M urea, pH 7.4. One tablet of the protease inhibitor cocktail (Roche, Mannheim, Germany) was freshly added to 10 ml of RIPA. Cell lysates were microcentrifuged at 14,000 rpm for 20 min at 4°C. Supernatants were collected and stored at –80°C. Protein concentration in each sample was determined by the BCA protein assay reagents (PIERCE, Rockford, IL). Cell lysates containing equal amounts of protein (25 or 50 μg) were boiled in an SDS sample buffer and analyzed by SDS–PAGE. The separated proteins in the gel were then transferred 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 such as a rabbit anti-LO (1:1,000), a rabbit anti-collagen type I (1:5,000), a goat anti-elastin (1:1,000), a mouse anti-MT (DAKO-MT, E-9, 1:500), or a rabbit anti--GCS (Neomarkers, Fremont, CA, 1:1000), washed with Tris-buffered saline with 0.1% Tween-20 three times each for 5 min and then incubated with the corresponding secondary antibody (i.e., anti-rabbit, anti-goat or anti-mouse IgG) conjugated with horseradish peroxidase (1:2000, Santa Cruz Biotech., Santa Cruz, CA) 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.
Reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was extracted from control and CSC-treated cells using a TRIzol Reagent Kit (Invitrogen, Carlsbad, CA). The mRNA expression of LO, MT-II, and -GCS was evaluated by RT-PCR as described (Chen et al., 2004). Briefly, 500 pg to 500 ng of total RNA for MT-II, LO, and -GCS were converted to the cDNA, which was then amplified by using the SuperScript One-Step RT-PCR with Platinum Taq Kit (Invitrogen, Carlsbad, CA) under the following conditions: reverse-transcript at 50°C for 30 min and predenaturation at 94°C for 2 min, denaturation at 94°C for 30 sec, annealing at 58°C (for MT-II) or 60°C (for LO and -GCS) for 30 sec, and extension at 72°C for 30 sec (for MT-II), 90 sec (for LO), or 2 min (for -GCS) for a total of 35 repetitive cycles. Final extension was performed at 72°C for 5–10 min. The primers used were 5'-GATGGTACCCTCGAGATGGACCCCAACTGCTCCTGTGCCACAGATGGATC-3' (Forward) and 5'-GATAAGCTTTCTAGATCAGGCGCAGCAGCTGCACTTGTCCGAAGCCTCTTT-3' (Reverse) for MT-II, 5'-GATGGATCCTCTAGAATGCGTTTCGCCTGGACCGTGCTCTTTCTGGG-3' (Forward) and 5'-GATCTCGAGGATATCCTAATACGGTGAAATGGTGCAGCCTGAGGCATAGGC-3' (Reverse) for LO as well as 5'-GATGGTACCCTCGAGATGGGGCTGCTGTCCCAAGGCTCGCCACTGAGCTGGGAA-3' (Forward) and 5'-GATAAGCTTTCTAGACTAGTCTGAAGGGTCGCTTTTACCTCCACTGTA-3' (Reverse) for -GCS. Glyceradehyde 3-phosphate dehydrogenase (GAPDH) was amplified in parallel as an internal control. The primers of GAPDH were 5'-GACTCTACCCACGGCAA-3' (Forward) and 5'-GGATGACCTTGCCCACA-3' (Reverse). Such PCR products as the 216 bp for MT-II, the 1.3 kb for LO, the 1.8 kb for -GCS, and the 500 bp for GAPDH were respectively separated on agarose gels, stained with ethidium bromide, and visualized on a UV transilluminator. cDNA bands were quantitated with 1 D Scan analysis as described above.
LO activity assay. Growth-arrested RFL6 cells in 0.3% FBS/DMEM were exposed to CSC at indicated doses for 24 h. The conditioned medium was collected and assayed for LO activity using a standard protocol against a recombinant human tropoelastin substrate labeled with 3H-lysine as described (Li et al., 1995). In a typical assay, samples (e.g., 700 μl conditioned medium) were incubated with 375,000 dpm of tritiated substrate for 3 h at 37°C in a 0.15 M NaCl/0.1 M sodium borate buffer, pH 8.0, in a total volume of 750 μl in the presence or absence of 0.5 mM -aminopropionitrile, an active site inhibitor of LO. Tritiated water released during the incubation was isolated by vacuum distillation and counted by liquid scintillation spectrometry. Enzyme activities were normalized to total cell protein.
Assay for total cellular GSH. Total cellular GSH was determined by the Tietze enzymatic assay (1969) as previously described (Li et al., 1994). Briefly, cells were rinsed twice with cold PBS, gently scraped into PBS, and centrifuged. The cell pellets were lysed in 0.2% Triton X-100 in PBS. The protein was precipitated by addition of cold 50% TCA to a final concentration of 2.5%, followed by centrifugation in a microcentrifuge. The supernatant was then assayed for total cellular GSH (including the reduced form, GSH, and the oxidized form, GSSG) by measuring the change in color of the reaction mixture at 412 nm in the presence of GSH reductase and nicotinamide adenine dinucleotide phosphate as described (Li et al., 1994). A standard curve for GSH was always generated with each assay, and the results are expressed as nanogram GSH per milligram of total cell protein.
Statistical analysis. In LO activity and total cellular GSH assays, data are presented as mean ± standard deviation (SD) of three separate experiments in which each control or variable was assessed in triplicate dishes. A one-way analysis of variance, followed by Dunnett test was performed. Differences were considered significant at p < 0.05.
RESULTS
CSC-Mediated Downregulation of LO at the mRNA, Protein, and Catalytic Levels in Cultured RFL6 Cells
To characterize the effects of CSC on the expression of LO, growth-arrested RFL6 cells were treated with CSC at the indicated doses (0–120 μg/ml) for 24 h, followed by assays for LO mRNA, protein, and catalytic activities as described in Materials and Methods. To assess LO mRNA expression by RT-PCR, equal amounts of total RNA isolated from control and treated cells were added to the reverse-transcription reaction mixture. Total cDNA produced by the reverse-transcription reaction and PCR amplification was evaluated as levels of transcripts. As shown (Fig. 1A), cells exposed to CSC exhibited dose-dependent decreases in levels of LO cDNA (1.3 kb) in comparison to the internal control, GAPDH cDNA (500 bp). One D Scan EX analysis revealed that CSC at 40, 80, and 120 μg/ml reduced LO cDNA to 58, 37, and 32%, respectively, of the control without CSC treatment, indicating CSC inhibition of LO mRNA expression. Furthermore, Western blot analysis indicated that CSC decreased levels of all LO protein species, including the 46 kDa preproenzyme, the 50 kDa proenzyme, and the extracellular, but cell membrane-associated 32 kDa mature enzyme, in a dose-dependent manner (Fig. 1B). Notably, stronger CSC inhibition occurred in LO species undergoing further downstream processing under the same conditions. For example, in cells exposed to CSC at 120 μg/ml, the 46 kDa preproenzyme, the 50 kDa proenzyme, and the 32 kDa mature enzyme were reduced to 61, 39, and 24%, respectively, of their corresponding controls, suggesting CSC perturbation of LO processing to its mature form in addition to inhibition of LO mRNA (Fig. 1A). Moreover, since LO activity was mainly present in the conditioned medium of fibrogenic cell cultures (Li et al., 1995), we also examined LO catalytic activities in conditioned media of control and CSC-treated cells by determining 3H-H2O2 release using 3H-tropoelastin as a substrate. As shown in Figure 1C, CSC at 20, 40, 80, and 120 μg/ml decreased LO activity in conditioned media of treated cells to 79, 60, 31, and 14% of the control (100% activity in control = 8,040 ± 851 dpm/mg total cell protein), respectively, with IC50 = 53 μg/ml. In comparison to the inhibitory effects of CSC on the protein level, higher doses of CSC (e.g., 80 and 120 μg/ml) further abolished the catalytic capacity of mature LO. For example, in the presence of 120 μg CSC/ml, cells retained mature LO protein amounting to 24% of the control but exhibiting only 14% of the control enzyme activity in the conditioned medium. Thus, the dose-dependent downregulation of LO in CSC-treated cells was considerably evident at mRNA, protein, and catalytic levels.
CSC Inhibition of Collagen Type I and Elastin Expression in Cultured RFL6 Cells
Collagen and elastin are substrates of LO. Collagen type I was used as a representative of the collagen protein family in this study. The protein profiles of collagen type I and tropoelastin were assayed in control and CSC-treated cells by Western blotting. As shown, a 24-h exposure of cells to CSC at 40–120 μg/ml inhibited the expression of collagen type I protein (Fig. 2), while prolonged treatment of cells with CSC at 100 μg/ml and 160 μg/ml for 1 week also markedly inhibited the expression of tropoelastin (Fig. 3) in cultured RFL6 cells. Under these treatment conditions with maximum doses of CSC, collagen type I and elastin were decreased by 50 and 67%, respectively. Thus, CSC also inhibited the expression of LO substrates, collagen type I and elastin, the major components of the lung ECM.
CSC Upregulation of MT at Protein and mRNA Levels in Cultured RFL6 Cells
MTs are small, inducible, cysteine-rich, metal-binding proteins functioning in metal detoxification, transport, and storage and anti-oxidative stress. Since CS contains heavy metals such as cadmium (Cd) and various oxidants (Hoffmann and Wynder, 1999), alterations in cellular MT expression may be an important parameter for cells exposed to CS. To test this possibility we examined MT at protein and mRNA levels in CSC-treated RFL6 cells. As shown (Fig. 4A), the basal level of MT-I and MT-II, major MT isoforms expressed in the lung (Hart et al., 1996) was, as expected, very low in control cells without CSC treatment. There were marked increases in cells treated with CSC at 40 μg, 80 μg, and 120 μg/ml amounting to 16-, 159-, and 456-fold of the control, respectively. Furthermore, RT-PCR results indicated that MT-II mRNA levels were elevated with increasing concentrations of CSC in cell cultures, as evidenced by enhanced levels of MT-II cDNA (216 bp) relative to levels of the internal control, GAPDH cDNA (500 bp) (Fig. 4 B). As analyzed by the 1 D Scan EX software, levels of MT-II mRNA in cells treated with 40, 80, and 120 μg CSC/ml reached 3.4-, 8.6-, and 9.2-fold of the control, respectively. These results show CSC induction of MT expression at protein and mRNA levels.
CSC Enhancement of Cellular GSH and Upregulation of -GCS at Protein and mRNA Levels in Cultured RFL6 Cells
GSH, a thiol-containing tripeptide, forms complexes with several metal ions, serving as a carrier for essential metal transport and for heavy metal detoxification (Freedman et al., 1989). To test CSC modulation of GSH expression, we examined cellular GSH levels and GSH synthetic enzyme -GCS at protein and mRNA levels in control and treated cells. The Tietze enzymatic assay indicated that exposure of cells to CSC at 20, 40, 80 and 120 μg/ml for 24 h increased cellular GSH levels to 161, 268, 315, and 331% of the control (100% GSH in the control = 1,157 ± 150 ng/mg total cell protein), respectively (Fig. 5A). Moreover, the parallel stimulatory effects of CSC on the expression of -GCS were also demonstrated. As revealed by Western blot and RT-PCR assays, both the -GCS protein (Fig. 5B) and its transcript (Fig. 5C) were elevated in CSC-treated cells with increasing concentrations of CSC in the media. For example, at 120 μg CSC/ml, the mRNA and protein levels of -GCS were increased to 210 and 280% of corresponding controls, respectively. Note that increased levels of -GCS mRNA in this study were reflected by elevated levels of the production of the -GCS cDNA (1.8 kb) in the reverse-transcription reaction. These results suggest CSC stimulation of cellular GSH biosynthesis catalyzed by -GCS.
Effects of GSH Elevation by GME on LO Protein and Catalytic Levels in Cultured RFL6 Cells
To assess the modulating effects of cellular GSH on LO expression, we have used a GSH delivery system; that is, treatment of RFL6 cells with GME since GME, but not GSH itself, can be easily transported into the cell. Following internalization, GME is hydrolyzed by esterases, releasing GSH intracellularly (Anderson, et al., 1985). To increase cellular GSH, growth-arrested RFL6 cells were exposed to GME at indicated doses for 1 h, followed by washing and incubating in the absence of GME for an additional 16 h. As shown (Fig. 6A), this 1-h pulse of GME at 2 and 4 mM elevated cellular GSH levels to 210 and 380%, respectively, of the control (100% GSH in the control = 962 ± 72 ng/mg total cell protein). This was associated with reductions of LO activities by 45 and 88%, respectively, in conditioned media of GME-treated cells as determined by the standardized 3H-tropoelastin assay (100% LO activity in the control = 6,486 ± 517 dpm/mg total cell protein). Furthermore, Western blot analysis indicated that exposure of cells to GME for 48 h markedly reduced levels of the 46 kDa LO preproenzyme, such that a 50% decrease occurred in cells treated with 4 mM GME in comparison to the control (Fig. 6B). These results directly point to downregulation of LO by elevation of cellular thiols.
DISCUSSION
In this study, we investigated the molecular and cellular events following CS exposure which may lead to the development of emphysema. Because of the central role of LO in morphogenesis and tissue repair of the lung ECM, we studied perturbations by CSC of the expression of LO, its collagen and elastin substrates, and its potential modulators, MT and GSH, using RFL6 cells as a model. We showed that CSC induced downregulation of LO expression at mRNA, protein, and catalytic levels concurrent with decreased levels of collagen type I and elastin, but upregulation of cellular thiols including MT and GSH as well as -GCS, a rate-limiting enzyme for GSH biosynthesis (Yan and Meister, 1990).
Results reported here show inhibition of LO by CSC at multiple levels. Decreased LO steady-state mRNA in treated cells implies that CSC may suppress initiation of gene transcription or enhance mRNA instability or both. Notably, our parallel studies have shown CSC-mediated inhibition of the relative rate of LO transcription and LO mRNA stability (Li et al., 2004). Thus, reduced levels of all LO protein species by CSC could result from decreases in LO mRNA. Furthermore, a stronger inhibition of downstream LO species, for example, the pro- and mature LO, in cells treated with CSC under the same conditions, suggests that several steps in LO translational and posttranslational processing may be susceptible to CSC insult. Moreover, CSC inhibition of LO catalytic activity in the conditioned medium in comparison to the cell associated 32 kDa species reflects that the mature enzyme could also be a target of CSC. Thus, CSC inhibited LO expression at the mRNA, protein, and catalytic levels. In view of essential biological functions of LO, such multilevel perturbations of LO would in turn inhibit the crosslinking of its substrates, collagen and elastin, favoring their destabilization, solubilization, and eventual degradation and interfering with their repair, finally leading to disruption of the lung ECM, key events characteristic in the development of emphysema.
Since collagen and elastin are major structural proteins of the lung ECM, their damage is implicated in the pathogenesis of emphysema (D'Armiento et al., 1992; Snider et al., 1986). It is of interest that the status of collagen and elastin in the ECM can regulate their own biosynthesis. For example, the N-terminal extension peptides cleaved from the maturing collagen molecules exerted a feedback inhibition on procollagen synthesis (Diegelmann and Peterkofsky, 1972). Addition of tropoelastin peptides to cell culture resulted in downregulation of tropoelastin mRNA (Jackson et al., 1991). Our data indicated that RFL6 cells exposed to CSC displayed markedly decreased levels of collagen type I and tropoelastin proteins. This may directly result from CSC insult to the expression of collagen and elastin genes at different levels. It should be pointed out that, since both collagen and elastin are substrates of LO, it is plausible to expect that LO functionality may also modulate the status of collagen and elastin in the ECM. As reported, lung injuries induced by administration of lathyrogens or Cu-deficient diets in experimental animals resembled panlobular emphysema in humans and occurred under the conditions of LO inhibition (Harris, 1986; O'Dell et al., 1978). Treatment of vascular smooth muscle cells with -aminopropionitrile, an active site inhibitor of LO, inhibited expression of tropoelastin mRNA (Jackson et al., 1991). Moreover, cells transfected with LO cDNA exhibited enhanced promoter activity of human collagen type III (Giampuzzi et al., 2000). Notably, here downregulation by CSC of collagen type I and elastin at the protein level was associated with marked reduction of LO at protein, mRNA, and catalytic levels in treated cells. This suggests that decreased levels of LO induced by CSC may play a key role in the downregulation of collagen and elastin by mechanisms that enhance instability of these proteins, leading to activation of the feedback inhibition described above.
CS contains more than 4000 different compounds including oxidants and toxic heavy metals (Hoffmann and Wynder, 1999). It produces 1014–1016 free radicals/puff, inducing an oxidative burden in the lung (Church and Pryor, 1985). CSC collected from one cigarette in accordance with the Federal Trade Commission smoking regime contains 69.3 ± 2.8 ng Cd, 6.0 ± 0.5 ng arsenic, and 42.0 ± 2.1 ng lead (Torrence et al., 2002). CS constitutes a major source of Cd exposure for smokers since tobacco leaves naturally accumulate Cd from soil (IARC, 1993). Pulmonary Cd levels reach 7.5-fold greater in smokers than in nonsmokers (Paakko et al., 1989). MT is a thiol-rich metalloprotein consisting of 61–62 amino acids, of which 20 are cysteines (Cys). Cys-SH groups provide metal-binding sites for MT. Exposure of cells to heavy metals such as Cd enhances the expression of MT (Kagi and Schaffer, 1988; Li et al., 1995). In this study, increased levels of MT II mRNA in CSC-treated cells indicated the activation of MT genes, resulting in elevated levels of MT protein in these cells (Figs. 4A and 4B). GSH, a thiol-containing tripeptide, accounts for 90% of total cellular nonprotein thiols (Meister, 1984). GSH can bind several metal ions mainly through its –SH group, thus functioning in essential metal transport and heavy metal scavenging. In addition, through the conversion of its reduced and oxidized forms catalyzed by GSH peroxidase and GSH reductase, GSH also eliminates reactive oxygen species (Meister, 1984). Data presented here show CSC stimulation of cellular GSH consistent with other reports (Rahman and MacNee, 1999). Moreover, our studies further demonstrate that activation of the -GCS gene contributed to enhancement of cellular GSH in CSC-treated cells (Figs. 5B and 5C). These results indicate that CSC is an effective inducer and/or stimulator of cellular MT and GSH. Elevation of such cellular thiols is expected to provide a critical defense mechanism against oxidative stress and heavy metal toxicity in the lungs of humans in response to CS exposure.
LO is a metalloenzyme requiring 1 mole of Cu (II) at its active site per mole of enzyme. Cu binding to the proenzyme 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). Other divalent metals such as Cd, Fe, Co, Zn, and Ni cannot replace Cu as a cofactor for LO (Gacheru et al., 1990). Dietary deprivation of Cu in animals induced lathyritic injuries manifested by reduction of elastin deposition in the lung due to suppression of LO activity (Harris, 1986; O'Dell et al., 1978; Soskel et al., 1984). Thiol-containing MT and GSH are critical modulators for Cu metabolism (Brouwer et al., 1993; Richards, 1989). MT can chelate 11–12 Cu molecules/mole of protein (Richards, 1989) which are in the cuprous state [Cu(I)]. MT has a higher affinity for Cu than Cd since Cd selectively displaced Zn but not Cu in native calf liver MT, which binds 4 moles of Zn and 3 moles of Cu per mole of MT (Briggs and Armitage, 1982). GSH can form a complex with Cu-MT, providing the biological basis for Cu transfer between these two cellular thiols (Brouwer et al., 1993). Reduction of Cu(II) to Cu(I) by GSH is thought to be a preliminary step in cellular transport of Cu. Our studies showed markedly elevated levels of MT and GSH in association with considerably decreased levels of LO in CSC-treated RFL6 cells. In light of the function of MT and GSH in Cu metabolism, it is expected that CSC-induced elevation of MT and GSH in treated cells may largely trap cellular Cu, thus limiting its biological availability for LO. This would be expected to lead to inhibition of catalytic activity of an enzyme that requires Cu as a cofactor. However, the precise role of Cu in the mechanism of action of LO remains to be understood (Kagan and Li, 2003). Notably, using isotope labeling we have demonstrated that, in MT-overexpressing RFL6 cells following long-term Cd exposure, radioactivity of 64Cu bound to the LO fraction amounted to only 9% of the control as compared to 1,400% of the control of 64Cu associated with the MT fraction (control = 64Cu radioactivity associated with the LO or MT fraction isolated from cells without Cd treatment) (Data not shown).
Direct correlation of elevated levels of cellular thiols with downregulation of LO in CSC-treated cells came from experiments in which RFL6 cells were exposed to GME, a GSH delivery system (Fig. 6). Clearly, elevation of cellular GSH was associated with reduction of LO activity in conditioned media of GME-treated cells. Moreover, Western blot analysis further indicated that exposure of cells to GME at 4 mM for 2 days markedly inhibited LO protein expression, especially the 46 kDa species. These results provide evidence directly linking the homeostasis of cellular thiols to the expression and regulation of LO in RFL6 cells.
In brief, our studies have revealed important cellular events elicited by CSC. CSC upregulated cellular thiols such as GSH and MT, but downregulated LO expression at the mRNA, protein, and catalytic levels, leading to destabilization of its substrates, collagen and elastin, the major components of the lung ECM critically involved in the pathogenesis of emphysema.
ACKNOWLEDGMENTS
This work was supported by research grants from the Philip Morris External Research Program and NIH R01-ES 11340.
NOTES
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ABSTRACT
Lysyl oxidase (LO), a copper-dependent enzyme, plays a critical role in the formation and repair of the extracellular matrix (ECM) by catalyzing the crosslinking of elastin and collagen. To better understand mechanisms of cigarette smoke (CS)-induced emphysema, we examined changes in LO and its substrates, i.e., elastin and collagen type I, the major components of cellular thiols, i.e., metallothionein (MT) and glutathione (GSH), and gamma-glutamylcysteine synthetase (-GCS), a key enzyme for GSH biosynthesis, in cigarette smoke condensate (CSC)-treated rat fetal lung fibroblasts (RFL6). Exposure of RFL6 cells to CSC decreased levels of LO catalytic activity, mRNA, and protein, i.e., the 46 kDa preproenzyme, the 50 kDa proenzyme and the 32 kDa mature enzyme in a dose-dependent manner. In addition, CSC also inhibited the expression of collagen type I and elastin, substrates of LO and important components of the lung ECM. Meanwhile, cellular thiols including MT and GSH as well as -GCS were markedly upregulated in CSC-treated cells. To evaluate modulation of LO expression by cellular thiols, we further examined the effect of increased levels of GSH on LO expression at protein and catalytic levels. Interestingly, exposure of cells to glutathione monoethyl ester, a GSH delivery system, effectively elevated cellular GSH levels and induced a dose-dependent decrease in levels of the protein species and catalytic activity of LO. These results suggest that upregulation by CSC of cellular thiols may play an important role in the downregulation of LO and subsequently destabilization of the lung ECM in CS-induced emphysema.
Key Words: cigarette smoke condensate; lysyl oxidase; glutathione; metallothionein; collagen type I; elastin.
INTRODUCTION
Lysyl oxidase (LO) (E.C. 1. 4. 3.13), a copper (Cu)-dependent amine oxidase, is initially synthesized by fibrogenic cells such as fibroblasts and vascular smooth muscle cells as a 46 kDa preproenzyme with N-terminal signal sequences which direct nascent proteins into the lumen of the rough endoplasmic reticulum. The proprotein resulting from cleavage of the N-terminal signal peptide undergoes N-linked glycosylation in the endoplasmic reticulum and the Golgi apparatus, and is then extracellularly secreted via secretory vesicles as a 50 kDa proenzyme. In the extracellular matrix (ECM), the 50 kDa proLO is further processed by proteolysis into the enzymatically active 32 kDa mature species (Trackman et al., 1992). LO catalyzes the covalent crosslinking of elastin and collagen by oxidizing peptidyl lysines in these proteins to form peptidyl -aminoadipic--semialdehyde. These aldehyde residues can spontaneously condense with neighboring peptidyl lysines or peptidyl aldehyde, leading to the formation of insoluble aggregates found in fibrillar collagen and elastin, thus stabilizing the ECM (Kagan and Li, 2003). Therefore, LO plays a critical role in lung morphogenesis and tissue repair.
There is increasing evidence that, in addition to the crosslinking of elastin and collagen in the ECM, LO also exhibits other biological functions. For example, expression of transfected, sense LO cDNA in cells transformed by p21 Ha-Ras suppresses Ha-Ras-induced tumorigenesis, indicating a Ras-suppressor effect of LO (Kenyon et al., 1991). The 32 kDa mature LO purified from bovine aorta displays strong chemotactic activities for monocytes and vascular smooth muscle cells (Lazarus et al., 1995; Li et al., 2000). LO and its oxidized substrates exist within the nuclei of cultured vascular smooth muscle cells and 3T3 fibroblasts (Li et al., 1997, 2002). Recent studies have indicated that LO oxidizes basic fibroblast growth factor and inactivates its mitogenic potential (Li et al., 2003).
Perturbation of LO expression is involved in human diseases. Increased LO activity is associated with fibrotic diseases such as lung and liver fibrosis and atherosclerosis, whereas decreased LO activity is associated with disorders of Cu metabolism like Menkes syndrome (Kagan and Li, 2003).
Long-term exposure of humans to cigarette smoke (CS) can result in emphysema. Most studies of emphysema pathogenesis have focused on the "elastase-antielastase imbalance" hypothesis (Snider, et al., 1986). Unfortunately, this hypothesis cannot explain all aspects of the emphysema-associated pathology. For example, the CS-induced emphysematous lung in guinea pigs exhibited breakdown of collagen without significant changes in elastin (Wright and Churg, 1995). Furthermore, transgenic mice that over-express the gene for human collagenase developed emphysema of early onset (D'Armiento et al., 1992). These findings suggest that the ‘elastase-antielastase imbalance’ is not the only mechanism for emphysema pathogenesis. Thus, the molecular basis for CS-induced emphysema remains unclear and requires further investigation.
Emphysema is currently defined as ‘a condition of the lung characterized by abnormal enlargement of the airspaces with destruction of the walls’ (Snider et al., 1986). The critical role of LO in emphysema development was reflected in the disruption of the lung structure in chicks, rats, and hamsters following diet-induced deficiency of Cu, a cofactor of LO (Harris, 1986; O'Dell et al., 1978; Soskel et al., 1984). These findings provide evidence that LO deficiency is associated with the pathogenesis of emphysema.
To better understand the role of LO in CS-induced emphysema, we have examined changes in LO at mRNA, protein, and catalytic levels as well as its substrates, collagen type I and elastin, and changes in key components of lung defenses against oxidants and heavy metals, i.e., cellular thiols such as glutathione (GSH) and metallothionein (MT). In the present study, we report that cigarette smoke condensate (CSC), the particulate matter of CS, inhibits the expression of LO and its substrates, collagen and elastin, but enhances the levels of cellular thiols, MT and GSH, as well as gamma-glutamylcysteine synthetase (-GCS), a key enzyme for GSH biosynthesis (Yan and Meister, 1990), in cultured rat fetal lung fibroblasts (RFL6).
MATERIALS AND METHODS
Materials. CSC was purchased from Murty Pharmaceutical Inc. (Lexington, KY). It was prepared using a Phipps-Bird 20-channel smoking machine designed for Federal Trade Commission testing. Kentucky standard cigarettes 1R3F (University of Kentucky, KY) were smoked, and the particulate matter was trapped onto filters. The amount of CSC obtained was determined by weight increase of the filter. CSC was dissolved from the filter in dimethyl sulfoxide by sonication to yield 4% solution. The average yield of CSC was 21 mg per cigarette including 1.3 mg nicotine. Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were ordered from GIBCO (Grand Island, NY). Glutathione monoethyl ester (GME) and other biochemicals were obtained from SIGMA (St. Louis, MO).
Cell culture and treatment. The fibroblastic RFL6 cell line derived from the fetal lung tissue of normal Sprague-Dawley rats was obtained from ATCC. Since RFL6 cells display the key characteristics of the pulmonary fibroblasts in expressing collagen type I, tropoelastin, and LO as shown below, they are an ideal model for this study. Cells were regularly maintained in DMEM containing 10% FBS. Cells were seeded at 8 x 104/ml in 100-mm dishes with 10 ml complete medium and incubated for 24 h. To standardize experimental conditions, cells were growth-arrested and synchronized at the G1 phase by incubation in 0.3% FBS/DMEM for 72 h, followed by changing to a fresh 0.3% FBS/DMEM and incubation for another 24 h before experiments (Li et al., 1995). Cells were treated for 24 h with CSC at final concentrations ranging from 0 to 120 μg/ml, unless otherwise indicated, then processed for further analysis. Control cells were incubated in the presence of vehicle only.
Western blot analysis. Control and treated cells were washed and lysed in the RIPA buffer composed of 1x PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 2 M urea, pH 7.4. One tablet of the protease inhibitor cocktail (Roche, Mannheim, Germany) was freshly added to 10 ml of RIPA. Cell lysates were microcentrifuged at 14,000 rpm for 20 min at 4°C. Supernatants were collected and stored at –80°C. Protein concentration in each sample was determined by the BCA protein assay reagents (PIERCE, Rockford, IL). Cell lysates containing equal amounts of protein (25 or 50 μg) were boiled in an SDS sample buffer and analyzed by SDS–PAGE. The separated proteins in the gel were then transferred 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 such as a rabbit anti-LO (1:1,000), a rabbit anti-collagen type I (1:5,000), a goat anti-elastin (1:1,000), a mouse anti-MT (DAKO-MT, E-9, 1:500), or a rabbit anti--GCS (Neomarkers, Fremont, CA, 1:1000), washed with Tris-buffered saline with 0.1% Tween-20 three times each for 5 min and then incubated with the corresponding secondary antibody (i.e., anti-rabbit, anti-goat or anti-mouse IgG) conjugated with horseradish peroxidase (1:2000, Santa Cruz Biotech., Santa Cruz, CA) 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.
Reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was extracted from control and CSC-treated cells using a TRIzol Reagent Kit (Invitrogen, Carlsbad, CA). The mRNA expression of LO, MT-II, and -GCS was evaluated by RT-PCR as described (Chen et al., 2004). Briefly, 500 pg to 500 ng of total RNA for MT-II, LO, and -GCS were converted to the cDNA, which was then amplified by using the SuperScript One-Step RT-PCR with Platinum Taq Kit (Invitrogen, Carlsbad, CA) under the following conditions: reverse-transcript at 50°C for 30 min and predenaturation at 94°C for 2 min, denaturation at 94°C for 30 sec, annealing at 58°C (for MT-II) or 60°C (for LO and -GCS) for 30 sec, and extension at 72°C for 30 sec (for MT-II), 90 sec (for LO), or 2 min (for -GCS) for a total of 35 repetitive cycles. Final extension was performed at 72°C for 5–10 min. The primers used were 5'-GATGGTACCCTCGAGATGGACCCCAACTGCTCCTGTGCCACAGATGGATC-3' (Forward) and 5'-GATAAGCTTTCTAGATCAGGCGCAGCAGCTGCACTTGTCCGAAGCCTCTTT-3' (Reverse) for MT-II, 5'-GATGGATCCTCTAGAATGCGTTTCGCCTGGACCGTGCTCTTTCTGGG-3' (Forward) and 5'-GATCTCGAGGATATCCTAATACGGTGAAATGGTGCAGCCTGAGGCATAGGC-3' (Reverse) for LO as well as 5'-GATGGTACCCTCGAGATGGGGCTGCTGTCCCAAGGCTCGCCACTGAGCTGGGAA-3' (Forward) and 5'-GATAAGCTTTCTAGACTAGTCTGAAGGGTCGCTTTTACCTCCACTGTA-3' (Reverse) for -GCS. Glyceradehyde 3-phosphate dehydrogenase (GAPDH) was amplified in parallel as an internal control. The primers of GAPDH were 5'-GACTCTACCCACGGCAA-3' (Forward) and 5'-GGATGACCTTGCCCACA-3' (Reverse). Such PCR products as the 216 bp for MT-II, the 1.3 kb for LO, the 1.8 kb for -GCS, and the 500 bp for GAPDH were respectively separated on agarose gels, stained with ethidium bromide, and visualized on a UV transilluminator. cDNA bands were quantitated with 1 D Scan analysis as described above.
LO activity assay. Growth-arrested RFL6 cells in 0.3% FBS/DMEM were exposed to CSC at indicated doses for 24 h. The conditioned medium was collected and assayed for LO activity using a standard protocol against a recombinant human tropoelastin substrate labeled with 3H-lysine as described (Li et al., 1995). In a typical assay, samples (e.g., 700 μl conditioned medium) were incubated with 375,000 dpm of tritiated substrate for 3 h at 37°C in a 0.15 M NaCl/0.1 M sodium borate buffer, pH 8.0, in a total volume of 750 μl in the presence or absence of 0.5 mM -aminopropionitrile, an active site inhibitor of LO. Tritiated water released during the incubation was isolated by vacuum distillation and counted by liquid scintillation spectrometry. Enzyme activities were normalized to total cell protein.
Assay for total cellular GSH. Total cellular GSH was determined by the Tietze enzymatic assay (1969) as previously described (Li et al., 1994). Briefly, cells were rinsed twice with cold PBS, gently scraped into PBS, and centrifuged. The cell pellets were lysed in 0.2% Triton X-100 in PBS. The protein was precipitated by addition of cold 50% TCA to a final concentration of 2.5%, followed by centrifugation in a microcentrifuge. The supernatant was then assayed for total cellular GSH (including the reduced form, GSH, and the oxidized form, GSSG) by measuring the change in color of the reaction mixture at 412 nm in the presence of GSH reductase and nicotinamide adenine dinucleotide phosphate as described (Li et al., 1994). A standard curve for GSH was always generated with each assay, and the results are expressed as nanogram GSH per milligram of total cell protein.
Statistical analysis. In LO activity and total cellular GSH assays, data are presented as mean ± standard deviation (SD) of three separate experiments in which each control or variable was assessed in triplicate dishes. A one-way analysis of variance, followed by Dunnett test was performed. Differences were considered significant at p < 0.05.
RESULTS
CSC-Mediated Downregulation of LO at the mRNA, Protein, and Catalytic Levels in Cultured RFL6 Cells
To characterize the effects of CSC on the expression of LO, growth-arrested RFL6 cells were treated with CSC at the indicated doses (0–120 μg/ml) for 24 h, followed by assays for LO mRNA, protein, and catalytic activities as described in Materials and Methods. To assess LO mRNA expression by RT-PCR, equal amounts of total RNA isolated from control and treated cells were added to the reverse-transcription reaction mixture. Total cDNA produced by the reverse-transcription reaction and PCR amplification was evaluated as levels of transcripts. As shown (Fig. 1A), cells exposed to CSC exhibited dose-dependent decreases in levels of LO cDNA (1.3 kb) in comparison to the internal control, GAPDH cDNA (500 bp). One D Scan EX analysis revealed that CSC at 40, 80, and 120 μg/ml reduced LO cDNA to 58, 37, and 32%, respectively, of the control without CSC treatment, indicating CSC inhibition of LO mRNA expression. Furthermore, Western blot analysis indicated that CSC decreased levels of all LO protein species, including the 46 kDa preproenzyme, the 50 kDa proenzyme, and the extracellular, but cell membrane-associated 32 kDa mature enzyme, in a dose-dependent manner (Fig. 1B). Notably, stronger CSC inhibition occurred in LO species undergoing further downstream processing under the same conditions. For example, in cells exposed to CSC at 120 μg/ml, the 46 kDa preproenzyme, the 50 kDa proenzyme, and the 32 kDa mature enzyme were reduced to 61, 39, and 24%, respectively, of their corresponding controls, suggesting CSC perturbation of LO processing to its mature form in addition to inhibition of LO mRNA (Fig. 1A). Moreover, since LO activity was mainly present in the conditioned medium of fibrogenic cell cultures (Li et al., 1995), we also examined LO catalytic activities in conditioned media of control and CSC-treated cells by determining 3H-H2O2 release using 3H-tropoelastin as a substrate. As shown in Figure 1C, CSC at 20, 40, 80, and 120 μg/ml decreased LO activity in conditioned media of treated cells to 79, 60, 31, and 14% of the control (100% activity in control = 8,040 ± 851 dpm/mg total cell protein), respectively, with IC50 = 53 μg/ml. In comparison to the inhibitory effects of CSC on the protein level, higher doses of CSC (e.g., 80 and 120 μg/ml) further abolished the catalytic capacity of mature LO. For example, in the presence of 120 μg CSC/ml, cells retained mature LO protein amounting to 24% of the control but exhibiting only 14% of the control enzyme activity in the conditioned medium. Thus, the dose-dependent downregulation of LO in CSC-treated cells was considerably evident at mRNA, protein, and catalytic levels.
CSC Inhibition of Collagen Type I and Elastin Expression in Cultured RFL6 Cells
Collagen and elastin are substrates of LO. Collagen type I was used as a representative of the collagen protein family in this study. The protein profiles of collagen type I and tropoelastin were assayed in control and CSC-treated cells by Western blotting. As shown, a 24-h exposure of cells to CSC at 40–120 μg/ml inhibited the expression of collagen type I protein (Fig. 2), while prolonged treatment of cells with CSC at 100 μg/ml and 160 μg/ml for 1 week also markedly inhibited the expression of tropoelastin (Fig. 3) in cultured RFL6 cells. Under these treatment conditions with maximum doses of CSC, collagen type I and elastin were decreased by 50 and 67%, respectively. Thus, CSC also inhibited the expression of LO substrates, collagen type I and elastin, the major components of the lung ECM.
CSC Upregulation of MT at Protein and mRNA Levels in Cultured RFL6 Cells
MTs are small, inducible, cysteine-rich, metal-binding proteins functioning in metal detoxification, transport, and storage and anti-oxidative stress. Since CS contains heavy metals such as cadmium (Cd) and various oxidants (Hoffmann and Wynder, 1999), alterations in cellular MT expression may be an important parameter for cells exposed to CS. To test this possibility we examined MT at protein and mRNA levels in CSC-treated RFL6 cells. As shown (Fig. 4A), the basal level of MT-I and MT-II, major MT isoforms expressed in the lung (Hart et al., 1996) was, as expected, very low in control cells without CSC treatment. There were marked increases in cells treated with CSC at 40 μg, 80 μg, and 120 μg/ml amounting to 16-, 159-, and 456-fold of the control, respectively. Furthermore, RT-PCR results indicated that MT-II mRNA levels were elevated with increasing concentrations of CSC in cell cultures, as evidenced by enhanced levels of MT-II cDNA (216 bp) relative to levels of the internal control, GAPDH cDNA (500 bp) (Fig. 4 B). As analyzed by the 1 D Scan EX software, levels of MT-II mRNA in cells treated with 40, 80, and 120 μg CSC/ml reached 3.4-, 8.6-, and 9.2-fold of the control, respectively. These results show CSC induction of MT expression at protein and mRNA levels.
CSC Enhancement of Cellular GSH and Upregulation of -GCS at Protein and mRNA Levels in Cultured RFL6 Cells
GSH, a thiol-containing tripeptide, forms complexes with several metal ions, serving as a carrier for essential metal transport and for heavy metal detoxification (Freedman et al., 1989). To test CSC modulation of GSH expression, we examined cellular GSH levels and GSH synthetic enzyme -GCS at protein and mRNA levels in control and treated cells. The Tietze enzymatic assay indicated that exposure of cells to CSC at 20, 40, 80 and 120 μg/ml for 24 h increased cellular GSH levels to 161, 268, 315, and 331% of the control (100% GSH in the control = 1,157 ± 150 ng/mg total cell protein), respectively (Fig. 5A). Moreover, the parallel stimulatory effects of CSC on the expression of -GCS were also demonstrated. As revealed by Western blot and RT-PCR assays, both the -GCS protein (Fig. 5B) and its transcript (Fig. 5C) were elevated in CSC-treated cells with increasing concentrations of CSC in the media. For example, at 120 μg CSC/ml, the mRNA and protein levels of -GCS were increased to 210 and 280% of corresponding controls, respectively. Note that increased levels of -GCS mRNA in this study were reflected by elevated levels of the production of the -GCS cDNA (1.8 kb) in the reverse-transcription reaction. These results suggest CSC stimulation of cellular GSH biosynthesis catalyzed by -GCS.
Effects of GSH Elevation by GME on LO Protein and Catalytic Levels in Cultured RFL6 Cells
To assess the modulating effects of cellular GSH on LO expression, we have used a GSH delivery system; that is, treatment of RFL6 cells with GME since GME, but not GSH itself, can be easily transported into the cell. Following internalization, GME is hydrolyzed by esterases, releasing GSH intracellularly (Anderson, et al., 1985). To increase cellular GSH, growth-arrested RFL6 cells were exposed to GME at indicated doses for 1 h, followed by washing and incubating in the absence of GME for an additional 16 h. As shown (Fig. 6A), this 1-h pulse of GME at 2 and 4 mM elevated cellular GSH levels to 210 and 380%, respectively, of the control (100% GSH in the control = 962 ± 72 ng/mg total cell protein). This was associated with reductions of LO activities by 45 and 88%, respectively, in conditioned media of GME-treated cells as determined by the standardized 3H-tropoelastin assay (100% LO activity in the control = 6,486 ± 517 dpm/mg total cell protein). Furthermore, Western blot analysis indicated that exposure of cells to GME for 48 h markedly reduced levels of the 46 kDa LO preproenzyme, such that a 50% decrease occurred in cells treated with 4 mM GME in comparison to the control (Fig. 6B). These results directly point to downregulation of LO by elevation of cellular thiols.
DISCUSSION
In this study, we investigated the molecular and cellular events following CS exposure which may lead to the development of emphysema. Because of the central role of LO in morphogenesis and tissue repair of the lung ECM, we studied perturbations by CSC of the expression of LO, its collagen and elastin substrates, and its potential modulators, MT and GSH, using RFL6 cells as a model. We showed that CSC induced downregulation of LO expression at mRNA, protein, and catalytic levels concurrent with decreased levels of collagen type I and elastin, but upregulation of cellular thiols including MT and GSH as well as -GCS, a rate-limiting enzyme for GSH biosynthesis (Yan and Meister, 1990).
Results reported here show inhibition of LO by CSC at multiple levels. Decreased LO steady-state mRNA in treated cells implies that CSC may suppress initiation of gene transcription or enhance mRNA instability or both. Notably, our parallel studies have shown CSC-mediated inhibition of the relative rate of LO transcription and LO mRNA stability (Li et al., 2004). Thus, reduced levels of all LO protein species by CSC could result from decreases in LO mRNA. Furthermore, a stronger inhibition of downstream LO species, for example, the pro- and mature LO, in cells treated with CSC under the same conditions, suggests that several steps in LO translational and posttranslational processing may be susceptible to CSC insult. Moreover, CSC inhibition of LO catalytic activity in the conditioned medium in comparison to the cell associated 32 kDa species reflects that the mature enzyme could also be a target of CSC. Thus, CSC inhibited LO expression at the mRNA, protein, and catalytic levels. In view of essential biological functions of LO, such multilevel perturbations of LO would in turn inhibit the crosslinking of its substrates, collagen and elastin, favoring their destabilization, solubilization, and eventual degradation and interfering with their repair, finally leading to disruption of the lung ECM, key events characteristic in the development of emphysema.
Since collagen and elastin are major structural proteins of the lung ECM, their damage is implicated in the pathogenesis of emphysema (D'Armiento et al., 1992; Snider et al., 1986). It is of interest that the status of collagen and elastin in the ECM can regulate their own biosynthesis. For example, the N-terminal extension peptides cleaved from the maturing collagen molecules exerted a feedback inhibition on procollagen synthesis (Diegelmann and Peterkofsky, 1972). Addition of tropoelastin peptides to cell culture resulted in downregulation of tropoelastin mRNA (Jackson et al., 1991). Our data indicated that RFL6 cells exposed to CSC displayed markedly decreased levels of collagen type I and tropoelastin proteins. This may directly result from CSC insult to the expression of collagen and elastin genes at different levels. It should be pointed out that, since both collagen and elastin are substrates of LO, it is plausible to expect that LO functionality may also modulate the status of collagen and elastin in the ECM. As reported, lung injuries induced by administration of lathyrogens or Cu-deficient diets in experimental animals resembled panlobular emphysema in humans and occurred under the conditions of LO inhibition (Harris, 1986; O'Dell et al., 1978). Treatment of vascular smooth muscle cells with -aminopropionitrile, an active site inhibitor of LO, inhibited expression of tropoelastin mRNA (Jackson et al., 1991). Moreover, cells transfected with LO cDNA exhibited enhanced promoter activity of human collagen type III (Giampuzzi et al., 2000). Notably, here downregulation by CSC of collagen type I and elastin at the protein level was associated with marked reduction of LO at protein, mRNA, and catalytic levels in treated cells. This suggests that decreased levels of LO induced by CSC may play a key role in the downregulation of collagen and elastin by mechanisms that enhance instability of these proteins, leading to activation of the feedback inhibition described above.
CS contains more than 4000 different compounds including oxidants and toxic heavy metals (Hoffmann and Wynder, 1999). It produces 1014–1016 free radicals/puff, inducing an oxidative burden in the lung (Church and Pryor, 1985). CSC collected from one cigarette in accordance with the Federal Trade Commission smoking regime contains 69.3 ± 2.8 ng Cd, 6.0 ± 0.5 ng arsenic, and 42.0 ± 2.1 ng lead (Torrence et al., 2002). CS constitutes a major source of Cd exposure for smokers since tobacco leaves naturally accumulate Cd from soil (IARC, 1993). Pulmonary Cd levels reach 7.5-fold greater in smokers than in nonsmokers (Paakko et al., 1989). MT is a thiol-rich metalloprotein consisting of 61–62 amino acids, of which 20 are cysteines (Cys). Cys-SH groups provide metal-binding sites for MT. Exposure of cells to heavy metals such as Cd enhances the expression of MT (Kagi and Schaffer, 1988; Li et al., 1995). In this study, increased levels of MT II mRNA in CSC-treated cells indicated the activation of MT genes, resulting in elevated levels of MT protein in these cells (Figs. 4A and 4B). GSH, a thiol-containing tripeptide, accounts for 90% of total cellular nonprotein thiols (Meister, 1984). GSH can bind several metal ions mainly through its –SH group, thus functioning in essential metal transport and heavy metal scavenging. In addition, through the conversion of its reduced and oxidized forms catalyzed by GSH peroxidase and GSH reductase, GSH also eliminates reactive oxygen species (Meister, 1984). Data presented here show CSC stimulation of cellular GSH consistent with other reports (Rahman and MacNee, 1999). Moreover, our studies further demonstrate that activation of the -GCS gene contributed to enhancement of cellular GSH in CSC-treated cells (Figs. 5B and 5C). These results indicate that CSC is an effective inducer and/or stimulator of cellular MT and GSH. Elevation of such cellular thiols is expected to provide a critical defense mechanism against oxidative stress and heavy metal toxicity in the lungs of humans in response to CS exposure.
LO is a metalloenzyme requiring 1 mole of Cu (II) at its active site per mole of enzyme. Cu binding to the proenzyme 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). Other divalent metals such as Cd, Fe, Co, Zn, and Ni cannot replace Cu as a cofactor for LO (Gacheru et al., 1990). Dietary deprivation of Cu in animals induced lathyritic injuries manifested by reduction of elastin deposition in the lung due to suppression of LO activity (Harris, 1986; O'Dell et al., 1978; Soskel et al., 1984). Thiol-containing MT and GSH are critical modulators for Cu metabolism (Brouwer et al., 1993; Richards, 1989). MT can chelate 11–12 Cu molecules/mole of protein (Richards, 1989) which are in the cuprous state [Cu(I)]. MT has a higher affinity for Cu than Cd since Cd selectively displaced Zn but not Cu in native calf liver MT, which binds 4 moles of Zn and 3 moles of Cu per mole of MT (Briggs and Armitage, 1982). GSH can form a complex with Cu-MT, providing the biological basis for Cu transfer between these two cellular thiols (Brouwer et al., 1993). Reduction of Cu(II) to Cu(I) by GSH is thought to be a preliminary step in cellular transport of Cu. Our studies showed markedly elevated levels of MT and GSH in association with considerably decreased levels of LO in CSC-treated RFL6 cells. In light of the function of MT and GSH in Cu metabolism, it is expected that CSC-induced elevation of MT and GSH in treated cells may largely trap cellular Cu, thus limiting its biological availability for LO. This would be expected to lead to inhibition of catalytic activity of an enzyme that requires Cu as a cofactor. However, the precise role of Cu in the mechanism of action of LO remains to be understood (Kagan and Li, 2003). Notably, using isotope labeling we have demonstrated that, in MT-overexpressing RFL6 cells following long-term Cd exposure, radioactivity of 64Cu bound to the LO fraction amounted to only 9% of the control as compared to 1,400% of the control of 64Cu associated with the MT fraction (control = 64Cu radioactivity associated with the LO or MT fraction isolated from cells without Cd treatment) (Data not shown).
Direct correlation of elevated levels of cellular thiols with downregulation of LO in CSC-treated cells came from experiments in which RFL6 cells were exposed to GME, a GSH delivery system (Fig. 6). Clearly, elevation of cellular GSH was associated with reduction of LO activity in conditioned media of GME-treated cells. Moreover, Western blot analysis further indicated that exposure of cells to GME at 4 mM for 2 days markedly inhibited LO protein expression, especially the 46 kDa species. These results provide evidence directly linking the homeostasis of cellular thiols to the expression and regulation of LO in RFL6 cells.
In brief, our studies have revealed important cellular events elicited by CSC. CSC upregulated cellular thiols such as GSH and MT, but downregulated LO expression at the mRNA, protein, and catalytic levels, leading to destabilization of its substrates, collagen and elastin, the major components of the lung ECM critically involved in the pathogenesis of emphysema.
ACKNOWLEDGMENTS
This work was supported by research grants from the Philip Morris External Research Program and NIH R01-ES 11340.
NOTES
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