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Am J Physiol Endocrinol Metab 293: E62-E69, 2007. First published February 27, 2007; doi:10.1152/ajpendo.00053.2007
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Overexpression of cysteine dioxygenase reduces intracellular cysteine and glutathione pools in HepG2/C3A cells

John E. Dominy, Jr., Jesse Hwang, and Martha H. Stipanuk

Department of Nutritional Sciences, Cornell University, Ithaca, New York

Submitted 22 January 2007 ; accepted in final form 13 February 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cysteine levels are carefully regulated in mammals to balance metabolic needs against the potential for cytotoxicity. It has been postulated that one of the major regulators of intracellular cysteine levels in mammals is cysteine dioxygenase (CDO). Hepatic expression of this catabolic enzyme increases dramatically in response to increased cysteine availability and may therefore be part of a homeostatic response to shunt excess toxic cysteine to more benign metabolites such as sulfate or taurine. Direct experimental evidence, however, is lacking to support the hypothesis that CDO is capable of altering steady-state intracellular cysteine levels. In this study, we expressed either the wild-type (WT) or a catalytically inactivated mutant (H86A) isoform of CDO in HepG2/C3A cells (which do not express endogenous CDO protein) and cultured them in different concentrations of extracellular cysteine. WT CDO, but not H86A CDO, was capable of reducing intracellular cysteine levels in cells incubated in physiologically relevant concentrations of cysteine. WT CDO also decreased the glutathione pool and potentiated the toxicity of CdCl2. These results demonstrate that CDO is capable of altering intracellular cysteine levels as well as glutathione levels.

sulfur metabolism; taurine; hepatoma; liver

WHAT MECHANISMS DO MAMMALS USE to regulate intracellular cysteine levels? The importance of this physiological question is underscored by the fact that mammals must strike a delicate balance when it comes to maintaining cysteine homeostasis. On one side of the balance, cysteine levels must be kept high enough to meet several fundamental processes of cellular function. Cysteine, like the other common amino acids, is used for protein synthesis. It is also used as a precursor for the synthesis of hypotaurine, taurine, inorganic sulfate, and hydrogen sulfide. Its sulfhydryl side chain forms the active moiety of coenzyme A, an acyl group carrier required for many metabolic processes, as well as of glutathione, the principal mammalian cellular thiol and redox buffer. Indeed, it is the sulfhydryl-containing side chain of cysteine that forms the cornerstone of many redox reactions in the cell.

On the other side of the balance, cysteine concentrations must also be kept below the threshold of cytotoxicity. Excess cysteine has been shown to be toxic in several animal models (5, 23, 24). In humans, associations have been shown between chronically high levels of cysteine and the development of rheumatoid arthritis (4), Parkinson's disease (14), Alzheimer's disease (14), and adverse pregnancy outcomes (10).

One of the major regulators of intracellular cysteine levels is thought to be the enzyme cysteine dioxygenase (CDO, EC 1.13.11.20 [EC] ). CDO catalyzes the oxidation of cysteine to cysteine sulfinic acid and constitutes the first step in the pathways leading to either sulfate/pyruvate or hypotaurine/taurine synthesis (Fig. 1). Claims for the ability of CDO to regulate intracellular cysteine levels have been predicated entirely on associative kinetics between cysteine and CDO levels in rat liver, the organ with the highest constitutive expression of CDO (8, 20, 22). In the strongest reported association, rats were adapted to a low-protein (i.e., low sulfur amino acid) diet and then switched to a high-protein (i.e., high sulfur amino acid) diet. The switch in diet caused hepatic cysteine levels to undergo an initial threefold increase that eventually returned to fasting levels within 12 h (22). The decrease in intracellular cysteine levels corresponded with a large increase (~25-fold) in the expression of hepatic CDO between 3 and 12 h. This change in CDO level occurs principally because of posttranslational alterations in the amount of CDO protein. Cysteine significantly increases the half-life of CDO by attenuating the rate at which the enzyme is ubiquitinated and subsequently degraded (9, 31). This regulatory mechanism may thus facilitate the degradation of cysteine when intracellular levels become too high and conserve intracellular cysteine for other metabolic pathways when intracellular levels become depleted.


Figure 1
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  		Fig. 1. Diagram of the position of cysteine dioxygenase within the major pathways of cysteine metabolism in mammals. Cysteine dioxygenase (CDO) catalyzes the irreversible conversion of cysteine to cysteine sulfinic acid. This is the first step in the production of hypotaurine/taurine and pyruvate/sulfate from cysteine.

 
Despite the clear association between the level of cysteine availability and CDO expression, there have been no reports of experiments designed to directly test whether CDO is capable of altering steady-state intracellular cysteine levels. Assessing this issue is important from both a biochemical and physiological perspective. From a biochemical standpoint, experiments in metabolic control analysis have shown that there are many instances wherein a change in enzyme concentration has only a minimal effect on substrate or metabolite concentration in the cell (11). A change in CDO expression therefore may have no impact at all on intracellular cysteine levels. From a physiological standpoint, understanding the contribution of CDO to the regulation of intracellular cysteine is underscored by two key observations. The first, as mentioned above, is the dramatic change in hepatic CDO levels that occurs in response to dietary cysteine intake. The second observation comes from the collective reports of differential regulation of CDO gene expression under conditions such as cellular differentiation, where it is not immediately clear if cellular cysteine availability is changing (1, 6, 7, 16, 18). The expression of CDO under these conditions could play an important role in altering the local redox environment.

The purpose of this study was therefore to directly evaluate the ability of CDO to affect intracellular cysteine levels. We achieved this by transfecting either catalytically active or inactive isoforms of CDO in HepG2/C3A cells, a human hepatoma cell line that does not express endogenous CDO protein. The effect of these transfections on total intracellular cysteine, glutathione, hypotaurine, and taurine pools was then measured. Our data suggest that catalytically competent CDO is indeed capable of altering intracellular cysteine levels as well as the total glutathione pool.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
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Cloning of CDO expression constructs. To transiently express CDO protein in HepG2/C3A cells, the open-reading frame of wild-type (WT) rat CDO (BAA11925 [GenBank] ) was subcloned into the NotI/BamHI restriction sites of the p3XFLAG-CMV-7.1 vector (Sigma). This expression vector added a 3.5-kDa 3xFLAG peptide sequence to the NH2 terminus of CDO, yielding a fusion protein with a final molecular mass of ~26 kDa. As an experimental control for the effect of protein overexpression on intracellular cysteine levels, a catalytically inactive mutant of CDO was used. Histidine residue 86 of rat CDO is one of three histidine residues essential for binding the active site iron cofactor of this protein. Mutation of any of these histidine residues to an alanine produces a catalytically inert protein (J. E. Dominy, C. R. Simmons, and M. H. Stipanuk, unpublished observation). Histidine-86 of WT CDO in the p3xFLAG-CMV-7.1 vector was mutated to an alanine using the QuikChange II Site-Directed Mutagenesis kit (Stratagene) and the primers (mutated nucleotide residues underlined) 5'-CATGGCAGCAGTATTGCCGATCACACGGACTC-3' and 5'-GAGTCCGTGTGATCGGCAATACTGCTGCCATG-3'. Both WT and mutant (H86A) expression constructs were sequence verified before use by the Cornell Biotechnology Resource Center.

Cell cultures and treatments. HepG2/C3A cells were obtained from the American Type Culture Collection. Cells were maintained in sulfur amino acid-free DMEM supplemented with 10% FBS, 2 mM glutamine, 1 mM sodium pyruvate, 0.1 mM L-methionine, 0.3 mM cysteine, and 1x MEM nonessential amino acid solution in a humidified atmosphere of 5% CO2-95% air at a temperature of 37°C.

Cells were seeded into 12-well tissue culture plates at a density of 2 x 105 cells/ml and allowed to attach for 24 h in the maintenance medium. Cells were then transfected with the CDO expression constructs at a confluency of ~95% with Lipofectamine 2000. Mock-transfected cells were transfected with the empty p3xFLAG-CMV-7.1 vector. Cultures were allowed to take up the plasmid-DNA complexes for 6 h, after which time the monolayers were washed two times with PBS, and the experimental medium was added. The experimental medium was analogous to the maintenance medium with the exception that the composition of cysteine was varied (0, 0.1, 0.3, or 1 mM) and 0.05 mM bathocuproine disulfonate was added to reduce the rate of cysteine autooxidation to cystine. Cells were incubated in the experimental medium for 24 h before harvesting.

For experiments involving the effect of CDO expression on sensitivity to CdCl2, a concentrated stock solution of CdCl2 was made in Milli-Q water and sterile filtered through a 0.2-µm membrane. A working solution was then prepared in the appropriate experimental culture medium. This medium was added to cells 6 h after transfection and remained on the cultures for 24 h until cell viability was assessed via the neutral red assay.

Western blot analysis. The expression of CDO was evaluated by Western blot analysis as previously described (9) using polyclonal rabbit anti-rat CDO antibody. Expression of cysteine sulfinic acid decarboxylase was examined by Western blotting using a polyclonal rabbit anti-rat cysteine sulfinic acid decarboxylase antibody as previously reported by Reymond et al. (27).

Measurement of total intracellular cysteine and glutathione/free thiol pools. After transfection (24 h), cells were washed three times with 0.5 ml ice-cold PBS, and then 0.25 ml of 5% (wt/vol) sulfosalicylic acid (SSA) was added to each well. Cells were scraped in an Eppendorf centrifuge tube and microfuged for 15 min at 15,000 g to pellet precipitated protein. The supernatant was removed and split into two aliquots, one for the analysis of total free cysteine and the other for the measurement of glutathione/total free thiols. The protein pellet was saved for protein quantification using the bicinchonic acid assay (Pierce). Protein values derived from the pellets were used to normalize the cysteine/glutathione data to nanomoles per total milligram protein.

Intracellular cysteine levels were measured by a modification of the method of Gaitonde (12). The pH of the SSA supernatant was adjusted to 8.3 using 10 N NaOH, and then 500 mM dithiothreitol (DTT) was added to yield a final concentration of 5 mM. Samples were reduced with DTT for 15 min at room temperature. Following reduction, 100 µl of sample were acidified with 0.l ml glacial acetic acid and then reacted with 0.1 ml acid ninhydrin reagent (250 mg ninhydrin dissolved in a mixture of 6 ml glacial acetic acid and 4 ml 12 N HCl) for 10 min at 100°C. After being heated, the sample mixture was cooled on ice for 3 min and then diluted to 1.0 ml with 95% ethanol. Sample absorbance was measured at 560 nm, and cysteine levels were quantified using cysteine hydrochloride standards (0–500 µM) dissolved in 5% SSA and processed in the same manner as the samples. Testing of this procedure by our laboratory has shown it to be a highly selective and sensitive means of detecting cysteine levels between 5 and 500 µM in cell extracts. Full wavelength scans showed no significant absorbance at 560 nm for ninhydrin-derivatized cysteamine, cystine, DTT, glutathione, homocysteine, or penicillamine (data not shown).

Glutathione levels were measured using 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) as previously described (33). Before the DTNB assay, 100 µl of sample were treated with 25 µl 0.5 M NaBH4 incubated for 1 h at 37°C to reduce all disulfides to free thiols. NaBH4 was quenched by the dropwise addition of 23.5 µl 1 N HCl, 23.5 µl acetone, and 18.75 µl 1 M Tris·Cl buffer (pH = 8.5). This procedure assays total acid-soluble thiols, of which glutathione has been shown to constitute the vast majority. Nevertheless, the assay can also detect acid-soluble cysteine. Cysteine values obtained from the ninhydrin assay were therefore subtracted from the DTNB data to more accurately estimate total free glutathione levels.

Quantification of hypotaurine/taurine levels. Intracellular hypotaurine and taurine levels were measured in 5% SSA extracts by HPLC as previously described (9).

Assay of cell viability. Cell viability of HepG2/C3A cultures was assessed by the neutral red assay (3). After exposure to CdCl2, cell monolayers were washed two times with PBS and then incubated for 2 h with neutral red dye (100 µg/ml; Acros Organics) dissolved in serum-free DMEM. Monolayers were then washed three times with PBS. Next, 1 ml of elution medium (50% ethanol/1% acetic acid) was added to liberate dye taken up by cells, and cells were placed on a horizontal rocker for 15 min in the dark to completely release the neutral red dye. Aliquots of the eluted mixture were removed, and absorbance at 540 nm was measured.

Statistical analyses. Data were analyzed by one-way ANOVA followed by comparison of means with Tukey's multiple-comparison test. Differences were accepted as significant at P ≤ 0.05. All values are presented as means ± SD.


   RESULTS
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 METHODS
 RESULTS
DISCUSSION
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Ectopic expression of CDO in C3A cells. HepG2/C3A cells do not express significant amounts of endogenous CDO protein; Western blots of cell lysates probed with anti-CDO antibody showed no detectable bands at the predicted molecular mass of 22.5 kDa (Fig. 2A). Nevertheless, HepG2/C3A cultures were able to successfully synthesize CDO protein upon transfection with the 3xFLAG-tagged constructs (Fig. 2A).


Figure 2
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  		Fig. 2. Western blot analysis of CDO expression in extracts from transfected HepG2/C3A cells. A: representative Western showing expression levels of wild-type (WT) 3xFLAG-tagged CDO and mutant His86->Ala (H86A) 3xFLAG-tagged CDO. Cells were transfected with the indicated construct and then incubated for 24 h in DMEM containing 0, 0.1, 0.3, or 1 mM cysteine. Total protein (50 µg) was loaded in each lane. Liver lysate (25 µg total protein) obtained from a rat on a low-protein diet (9) was also run on this gel to demonstrate the relative level of CDO expression attainable in the cell culture system vs. what is seen in vivo. The molecular mass of endogenous CDO protein is 23 kDa, and the molecular mass of the FLAG-tagged CDOs is 26 kDa. The absence of endogenous CDO in HepG2/C3A cells is obvious from the lack of a detectable 23-kDa band. B: densitometric analysis of 3xFLAG-tagged CDO expression as determined by Western blot results from 3 independent experiments. Density values are normalized by the average expression level of cells transfected with 3xFLAG-tagged WT-CDO and treated with 0.1 mM cysteine.

 
Previous reports have shown that steady-state CDO expression in primary rat hepatocyte cultures is sensitive to changes in cysteine concentration. An analogous regulation was seen in HepG2/C3A cells. Protein expression of WT CDO was increased by ~160% when extracellular cysteine levels were increased from 0 to 1 mM cysteine (Fig. 2B). Expression of the catalytically inactive H86A mutant, on the other hand, was not sensitive to changes in cysteine availability. Constitutive expression of the H86A mutant was approximately two times as high as WT in the absence of extracellular cysteine and remained at this level even when medium cysteine levels were increased to 1 mM.

Although we were successful in ectopically expressing CDO in our cell culture system, the absolute level of maximal expression for either of the CDO constructs was far less than what is normally found in vivo. The maximum amount of CDO that could be expressed in the HepG2/C3A cells was, on a per gram total protein basis, ~30% of the level found in the liver of a rat fed a low-protein diet for 12 h (Fig. 2A).

Catalytically active CDO significantly decreased intracellular cysteine levels. In cells transfected with the empty vector alone, intracellular cysteine levels increased in concert with the amount of cysteine added to the medium (Fig. 3). The response, however, was not linear. When extracellular cysteine was raised from 0 to 0.3 mM, intracellular cysteine levels in this treatment group increased from beneath the limits of detection to 7.3 nmol/mg protein. Intracellular cysteine levels rose less quickly, from 7.3 to 12 nmol/mg protein, when extracellular cysteine levels were further increased from 0.3 to 1 mM.


Figure 3
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  		Fig. 3. Intracellular cysteine levels of transfected HepG2/C3A cells. Cells were transfected with either empty vector (CON), WT 3x-FLAG tagged CDO, or H86A 3xFLAG-tagged CDO and then incubated for 24 h in DMEM containing 0, 0.1, 0.3, or 1 mM cysteine. Results are from 3–5 independent experiments. *P < 0.05 and **P < 0.01 vs. CON group.

 
Consistent with the hypothesis that CDO could affect intracellular cysteine levels, transfection with WT CDO significantly reduced the amount of intracellular cysteine relative to the empty vector control. The magnitude of the effect, however, was inversely related to the amount of cysteine present in the medium. At an extracellular cysteine level of 0.1 mM, transfection with WT CDO resulted in a 40% decrease in intracellular cysteine relative to control. The effect was diminished to a 23% reduction when the extracellular cysteine was increased to 0.3 mM. No significant change in intracellular cysteine was observed when an extracellular cysteine concentration of 1 mM was used.

Transfection with the catalytically inoperative H86A mutant produced no effect on intracellular cysteine levels regardless of the concentration of cysteine in the medium. This negative control clearly showed that the mere overexpression of protein was not able to significantly perturb steady-state cysteine levels.

CDO significantly reduced intracellular glutathione levels and increased susceptibility to CdCl2 toxicity. In liver, the synthesis of glutathione is frequently limited by the availability of cysteine (13). Similarly, it has been also shown for HepG2/C3A cells that changes in the level of intracellular cysteine are closely paralleled by changes in the level of glutathione (21). For these reasons, we proceeded to evaluate whether the decrease in intracellular cysteine induced by CDO expression was accompanied by a decrease in steady-state glutathione levels.

Corresponding with the significant decrease in intracellular cysteine levels, transfection with WT CDO produced a significant decrease in intracellular glutathione levels when cysteine concentrations in the medium were 0.1 and 0.3 mM (Fig 4). The magnitude of the decrease in glutathione, however, was less than that observed for cysteine: 25 vs. 40% at 0.1 mM cysteine and 20 vs. 23% at 0.3 mM cysteine. Glutathione levels were not altered by the expression of WT-CDO when medium cysteine concentrations were either 0 or 1 mM. They were also not affected by transfection with H86A CDO. These latter results are consistent with the failure of these treatments to alter intracellular cysteine levels.


Figure 4
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  		Fig. 4. Intracellular glutathione levels of transfected HepG2/C3A cells. Cells were transfected with either empty vector (CON), WT 3xFLAG-tagged CDO, or H86A 3xFLAG-tagged CDO and then incubated for 24 h in DMEM containing 0, 0.1, 0.3, or 1 mM cysteine. Results are from 3–5 independent experiments. *P < 0.05 vs. CON group.

 
To test whether the 20–25% declines in intracellular glutathione induced by CDO expression had any physiological significance, we subjected HepG2/C3A cells to a glutathione-dependent stressor in the form of the heavy metal cadmium (CdCl2). Resistance to cadmium-mediated cytoxicity in hepatocytes is known to be directly dependent on intracellular glutathione levels (15, 17, 26, 35). In cells transfected with the empty vector, sensitivity to CdCl2 was affected by the concentration of cysteine in the medium (Fig. 5). Higher amounts of cysteine in the medium conferred protection against CdCl2 toxicity up to a concentration of 0.3 mM. Further increasing the medium concentration of cysteine to 1 mM failed to provide any additional protection against CdCl2 toxicity. A similar pattern was seen for cells transfected with H86A CDO. Cells transfected with WT CDO, on the other hand, showed an enhanced sensitivity to CdCl2 (Fig. 5, B and C) that was limited to cells cultured in medium with cysteine levels of 0.1 and 0.3 mM, conditions under which CDO transfection was shown to significantly decrease cysteine and glutathione levels.


Figure 5
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  		Fig. 5. Cell viability 24 h after incubation with different levels of CdCl2. Medium used in this experiment was supplemented with 0 (A), 0.1 (B), 0.3 (C), or 1 (D) mM cysteine. The concentration of cadmium corresponding to the median cell survival is listed for each of the transfected cell groups. Results are from 3 independent experiments.

 
Transfection with CDO failed to alter steady-state intracellular hypotaurine/taurine levels. CDO catalyzes the first step in the pathway leading to the synthesis of hypotaurine and taurine. Cysteine sulfinic acid produced by CDO is decarboxylated to hypotaurine by cysteine sulfinic acid decarboxylase. Hypotaurine is then oxidized to taurine by an ill-defined mechanism. Although hepatic levels of hypotaurine and taurine are well correlated with the expression of CDO in vivo, the exact contribution of CDO to total pathway flux is not entirely clear. We were therefore interested in testing whether CDO expression in the HepG2/C3A cells would increase steady-state intracellular hypotaurine/taurine levels. Surprisingly, transfection with WT CDO produced no significant change in intracellular levels of either hypotaurine or taurine (Fig. 6A). Further investigation revealed that the inability of CDO expression to alter these two amino acids was most likely because of the lack of expression of cysteine sulfinic acid decarboxylase in HepG2/C3A cells (Fig. 6B).


Figure 6
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  		Fig. 6. A: measurement of intracellular hypotaurine and taurine levels in transfected HepG2/C3A cells incubated for 24 h in medium supplemented with 0.3 mM cysteine. Results were obtained from 3 independent experiments. B: Western blot of rat liver and HepG2/C3A cell lysate probing for the expression of cysteine sulfinic acid decarboxylase. Total protein loaded/lane is indicated in parentheses.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Before this study, evidence for the ability of CDO to change intracellular cysteine levels was indirectly derived from the positive correlation between hepatic CDO expression and cysteine availability. It was concluded through teleological reasoning that the changes in CDO expression were part of a homeostatic response to maintain constant intracellular cysteine levels in the face of fluctuations in extracellular supply. In this study, we have demonstrated that the expression of catalytically active CDO can directly alter intracellular cysteine levels. It would also appear that, by altering intracellular cysteine levels, CDO is capable of indirectly modulating the total glutathione pool.

The expression of CDO was able to reduce intracellular cysteine levels in HepG2/C3A cells, but the effect was dependent on the amount of extracellular cysteine to which the cultures were exposed. The largest effect of CDO on intracellular cysteine levels was seen when 100 µM cysteine was included in the medium. This extracellular concentration of cysteine is comparable to what is found in vivo: plasma total cysteine levels range from 80 to 200 µM, with an average of ~100 µM (30). When extracellular cysteine levels were increased >0.1 mM, however, the expression of CDO had a markedly reduced effect on intracellular cysteine levels. At these higher, supraphysiological levels of extracellular cysteine, it would appear that the rate of transport and intracellular accumulation of cysteine was much greater than the rate at which CDO was catabolizing cysteine. One factor that may have contributed to this phenomenon is the nonlinear rate at which CDO accumulated in the HepG2/C3A cells in response to changes in extracellular cysteine levels. Changing extracellular cysteine from 0 to 0.1 mM produced a 50% increase in the expression of CDO protein. Further increasing cysteine levels from 0.1 to 1 mM, however, resulted in an additional increase in CDO expression of only 40%. Why the rate of increase in CDO protein expression tapered at the supraphysiological levels of cysteine is not clear. Nevertheless, at physiological concentrations of extracellular cysteine, the capacity of CDO to decrease intracellular cysteine levels is consistent with the enzyme's presumed role in the liver as a means for disposing of excess cysteine. Moreover, we propose that the effect of CDO on intracellular cysteine levels in vivo is even more pronounced than what was seen in the HepG2/C3A cells because the absolute levels of CDO protein expression in vivo are substantially higher. As shown in Fig. 2A, the amount of 3xFLAG-tagged CDO expression that was achievable in the HepG2/C3A cells was ~30% of the amount of CDO endogenously expressed in the liver of a rat on a low-protein diet. Hepatic CDO levels in a rat fed a low-protein diet represent the lower end of the potential expression spectrum of this enzyme. Hepatic CDO levels, for instance, increase ~30-fold when a rat is switched from a low-protein to a high-protein diet (2).

Glutathione levels also appeared to change in response to CDO expression. This is perhaps not too surprising given that cysteine is frequently a limiting substrate in the synthesis of glutathione. As seen in the experiments with CdCl2, the CDO-induced perturbation of glutathione production could have a significant effect on the redox capacity of the cell. Interestingly, this would suggest that, under certain conditions, another biological role of CDO may be to decrease intracellular glutathione levels. This finding may help to explain the results of several reported microarray studies showing that the expression of CDO significantly increases during the differentiation of many tissue types (1, 6, 7, 16, 18). During the process of differentiation, the intracellular redox potential becomes progressively more oxidized (19, 25, 29). An increased expression of CDO during differentiation could facilitate the development of this oxidizing environment by decreasing the amount of cysteine and, in turn, limiting the amount of substrate available for glutathione synthesis.

Although WT CDO protein was stabilized by cysteine, consistent with what has been reported previously (9, 31), expression of the H86A CDO mutant protein did not appear to be affected by changes in cysteine. In striking contrast to WT CDO, H86A CDO showed a high level of expression, even when cells were depleted of intracellular cysteine. This result was not anticipated but served as a useful control to illustrate that ectopic expression of a protein by itself did not change intracellular cysteine levels. Why this mutant failed to be appropriately regulated is not known. It is possible that mutation of the iron-binding histidines changes the tertiary structure of CDO in such a way that it is no longer recognized by an ubiquitin ligase, resulting in the disruption of cysteine-sensitive ubiquitination and degradation. Additional experiments are needed to resolve this issue.

Within a comparative physiology framework, this study has also revealed that there are significant differences in how cysteine is metabolized between HepG2/C3A cultures and hepatocytes in vivo. When incubated with physiological levels of cysteine, HepG2/C3A cells showed intracellular cysteine levels that were almost an order of magnitude higher than what is typically found in the liver in vivo [2.4 nmol/mg protein in control HepG2/C3A cells vs. 0.2–0.8 nmol/mg protein in liver (9, 22)]. The underlying reason for the enormous discrepancy in steady-state levels of cysteine is not known. It is unlikely that the higher levels of cysteine are the result of enhanced transsulfuration flux since HepG2 cells lack the high-Km form of methionine adenosyltransferase (34) and show a limited ability to convert methionine to cysteine (21). One possible explanation, however, is that cysteine levels are elevated in the HepG2/C3A cells as part of a protective adaptation against the much higher oxygen tensions associated with cell culture systems. Consistent with this is the observation that cultured cells show an upregulation of cystine transport that increases with the length of culturing (32). Another explanation is that the accumulation of cysteine in the transformed cell line is a direct consequence of not expressing CDO protein. Interestingly, the lack of CDO expression is not simply limited to the HepG2/C3A cell line. We have conducted Western blots on several different hepatocyte cell lines (HepG2, AML12, and McARH7777) and have failed to find any detectable CDO protein (L. L. Hirschberger, J. E. Dominy, and M. H. Stipanuk, unpublished observations). Why these transformed cells have lost the ability to express CDO protein is an interesting biological question that deserves further exploration.

Another metabolic difference between HepG2/C3A cells and normal hepatocytes uncovered in this study is the ability to synthesize taurine from cysteine. In fact, the transformed cells have taurine levels that are on the lower end of what is normally seen in the liver [2.8 nmol/mg protein in HepG2/C3A cells vs. an average of 12.8 nmol/mg protein in hepatocytes in vivo] (9, 22). The inability to synthesize taurine from cysteine in the HepG2/C3A cells appears to be the result of not only the absence of CDO protein but also the absence of cysteine sulfinic acid decarboxylase expression.

In conclusion, our study provides the first definitive evidence that the expression of CDO can significantly decrease the level of intracellular cysteine. This reduction is also associated with a decrease in total glutathione levels, presumably through a simple substrate limitation mechanism. These observations expand the role of CDO beyond the production of sulfate and taurine to include one of intracellular redox regulation.


   GRANTS
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 ABSTRACT
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 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-056649 awarded to M. H. Stipanuk. J. E. Dominy, Jr. was supported by a graduate student fellowship from the National Science Foundation.


   ACKNOWLEDGMENTS
 
We thank Lawrence Hirschberger for technical assistance with measuring hypotaurine and taurine levels. We also thank Dr. Marcel Tappaz (Institut National de la Santé et de la Recherche Médicale, Lyon, France) for providing cysteine sulfinic acid decarboxylase antibody.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. H. Stipanuk, Div. of Nutritional Sciences, 227 Savage Hall, Cornell Univ., Ithaca, NY 14853 (e-mail: mhs6{at}cornell.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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  1. Albert Y, Whitehead J, Eldredge L, Carter J, Gao X, Tourtellotte WG. Transcriptional regulation of myotube fate specification and intrafusal muscle fiber morphogenesis. J Cell Biol 169: 257–268, 2005.[Abstract/Free Full Text]
  2. Bella DL, Hirschberger LL, Hosokawa Y, Stipanuk MH. Mechanisms involved in the regulation of key enzymes of cysteine metabolism in rat liver in vivo. Am J Physiol Endocrinol Metab 276: E326–E335, 1999.[Abstract/Free Full Text]
  3. Borenfreund E, Puerner JA. Toxicity determined in vitro by morphological alterations and neutral red absorption. Toxicol Lett 24: 119–124, 1985.[CrossRef][Web of Science][Medline]
  4. Bradley H, Gough A, Sokhi RS, Hassell A, Waring R, Emery P. Sulfate metabolism is abnormal in patients with rheumatoid arthritis. Confirmation by in vivo biochemical findings. J Rheumatol 21: 1192–1196, 1994.[Web of Science][Medline]
  5. Andine P, Orwar O, Jacobson I, Sandberg M, Hagberg H. Extracellular acidic sulfur-containing amino acids and gamma-glutamyl peptides in global ischemia: postischemic recovery of neuronal activity is paralleled by a tetrodotoxin-sensitive increase in cysteine sulfinate in the CA1 of the rat hippocampus. J Neurochem 57: 230–236, 1991.[CrossRef][Web of Science][Medline]
  6. Bujalska IJ, Quinkler M, Tomlinson JW, Montague CT, Smith DM, Stewart PM. Expression profiling of 11beta-hydroxysteroid dehydrogenase type-1 and glucocorticoid-target genes in subcutaneous and omental human preadipocytes. J Mol Endocrinol 37: 327–340, 2006.[Abstract/Free Full Text]
  7. Chen SS, Chen JF, Johnson PF, Muppala V, Lee YH. C/EBPbeta, when expressed from the C/ebpalpha gene locus, can functionally replace C/EBPalpha in liver but not in adipose tissue. Mol Cell Biol 20: 7292–7299, 2000.[Abstract/Free Full Text]
  8. Cresenzi CL, Lee JI, Stipanuk MH. Cysteine is the metabolic signal responsible for dietary regulation of hepatic cysteine dioxygenase and glutamate cysteine ligase in intact rats. J Nutr 133: 2697–2702, 2003.[Abstract/Free Full Text]
  9. Dominy JE Jr, Hirschberger LL, Coloso RM, Stipanuk MH. Regulation of cysteine dioxygenase degradation is mediated by intracellular cysteine levels and the ubiquitin-26 S proteasome system in the living rat. Biochem J 394: 267–273, 2006.[CrossRef][Web of Science][Medline]
  10. El-Khairy L, Vollset SE, Refsum H, Ueland PM. Plasma total cysteine, pregnancy complications, and adverse pregnancy outcomes: the Hordaland Homocysteine Study. Am J Clin Nutr 77: 467–472, 2003.[Abstract/Free Full Text]
  11. Fell DA. Metabolic control analysis: a survey of its theoretical and experimental development. Biochem J 286: 313–330, 1992.[Web of Science][Medline]
  12. Gaitonde MK. A spectrophotometric method for the direct determination of cysteine in the presence of other naturally occurring amino acids. Biochem J 104: 627–633, 1967.[Web of Science][Medline]
  13. Griffith OW. Biologic and pharmacologic regulation of mammalian glutathione synthesis. Free Radic Biol Med 27: 922–935, 1999.[CrossRef][Web of Science][Medline]
  14. Heafield MT, Fearn S, Steventon GB, Waring RH, Williams AC, Sturman SG. Plasma cysteine and sulphate levels in patients with motor neurone, Parkinson's and Alzheimer's disease. Neurosci Lett 110: 216–220, 1990.[CrossRef][Web of Science][Medline]
  15. Ikediobi CO, Badisa VL, Ayuk-Takem LT, Latinwo LM, West J. Response of antioxidant enzymes and redox metabolites to cadmium-induced oxidative stress in CRL-1439 normal rat liver cells. Int J Mol Med 14: 87–92, 2004.[Web of Science][Medline]
  16. Kalajzic I, Staal A, Yang WP, Wu Y, Johnson SE, Feyen JH, Krueger W, Maye P, Yu F, Zhao Y, Kuo L, Gupta RR, Achenie LE, Wang HW, Shin DG, Rowe DW. Expression profile of osteoblast lineage at defined stages of differentiation. J Biol Chem 280: 24618–24626, 2005.[Abstract/Free Full Text]
  17. Kim SC, Cho MK, Kim SG. Cadmium-induced non-apoptotic cell death mediated by oxidative stress under the condition of sulfhydryl deficiency. Toxicol Lett 144: 325–336, 2003.[CrossRef][Web of Science][Medline]
  18. Kim TY, Zhong S, Fields CR, Kim JH, Robertson KD. Epigenomic profiling reveals novel and frequent targets of aberrant DNA methylation-mediated silencing in malignant glioma. Cancer Res 66: 7490–7501, 2006.[Abstract/Free Full Text]
  19. Kirlin WG, Cai J, Thompson SA, Diaz D, Kavanagh TJ, Jones DP. Glutathione redox potential in response to differentiation and enzyme inducers. Free Radic Biol Med 27: 1208–1218, 1999.[CrossRef][Web of Science][Medline]
  20. Kwon YH, Stipanuk MH. Cysteine regulates expression of cysteine dioxygenase and gamma-glutamylcysteine synthetase in cultured rat hepatocytes. Am J Physiol Endocrinol Metab 280: E804–E815, 2001.[Abstract/Free Full Text]
  21. Lee JI, Kang J, Stipanuk MH. Differential regulation of glutamate-cysteine ligase subunit expression and increased holoenzyme formation in response to cysteine deprivation. Biochem J 393: 181–190, 2006.[CrossRef][Web of Science][Medline]
  22. Lee JI, Londono M, Hirschberger LL, Stipanuk MH. Regulation of cysteine dioxygenase and gamma-glutamylcysteine synthetase is associated with hepatic cysteine level. J Nutr Biochem 15: 112–122, 2004.[CrossRef][Web of Science][Medline]
  23. Lehmann A. Alterations in hippocampal extracellular amino acids and purine catabolites during limbic seizures induced by folate injections into the rabbit amygdala. Neuroscience 22: 573–578, 1987.[CrossRef][Web of Science][Medline]
  24. Lehmann A, Hagberg H, Orwar O, Sandberg M. Cysteine sulphinate and cysteate: mediators of cysteine toxicity in the neonatal rat brain? Eur J Neurosci 5: 1398–1412, 1993.[CrossRef][Web of Science][Medline]
  25. Nkabyo YS, Ziegler TR, Gu LH, Watson WH, Jones DP. Glutathione and thioredoxin redox during differentiation in human colon epithelial (Caco-2) cells. Am J Physiol Gastrointest Liver Physiol 283: G1352–G1359, 2002.[Abstract/Free Full Text]
  26. Pourahmad J, O'Brien PJ. A comparison of hepatocyte cytotoxic mechanisms for Cu2+ and Cd2+. Toxicology 143: 263–273, 2000.[CrossRef][Web of Science][Medline]
  27. Reymond I, Bitoun M, Levillain O, Tappaz M. Regional expression and histological localization of cysteine sulfinate decarboxylase mRNA in the rat kidney. J Histochem Cytochem 48: 1461–1468, 2000.[Abstract/Free Full Text]
  28. Smith J, Ladi E, Mayer-Proschel M, Noble M. Redox state is a central modulator of the balance between self-renewal and differentiation in a dividing glial precursor cell. Proc Natl Acad Sci USA 97: 10032–10037, 2000.[Abstract/Free Full Text]
  29. Stipanuk MH, Dominy JE Jr, Lee JI, Coloso RM. Mammalian cysteine metabolism: new insights into regulation of cysteine metabolism. J Nutr 136: 1652S–1659S, 2006.[Abstract/Free Full Text]
  30. Stipanuk MH, Hirschberger LL, Londono MP, Cresenzi CL, Yu AF. The ubiquitin-proteasome system is responsible for cysteine-responsive regulation of cysteine dioxygenase concentration in liver. Am J Physiol Endocrinol Metab 286: E439–E448, 2004.[Abstract/Free Full Text]
  31. Takada A, Bannai S. Transport of cystine in isolated rat hepatocytes in primary culture. J Biol Chem 259: 2441–2445, 1984.[Abstract/Free Full Text]
  32. Tietze F. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal Biochem 27: 502–522, 1969.[CrossRef][Web of Science][Medline]
  33. Torres L, Avila MA, Carretero MV, Latasa MU, Caballeria J, Lopez-Rodas G, Boukaba A, Lu SC, Franco L, Mato JM. Liver-specific methionine adenosyltransferase MAT1A gene expression is associated with a specific pattern of promoter methylation and histone acetylation: implications for MAT1A silencing during transformation. FASEB J 14: 95–102, 2000.[Abstract/Free Full Text]
  34. Xu J, Maki D, Stapleton SR. Mediation of cadmium-induced oxidative damage and glucose-6-phosphate dehydrogenase expression through glutathione depletion. J Biochem Mol Toxicol 17: 67–75, 2003.[CrossRef][Web of Science][Medline]



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