1Department of Environmental Health Sciences, School of Public Health, and 2Department of Medicine and Pathology and 3Department of Anesthesiology, School of Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294-0022
Submitted 15 July 2003 ; accepted in final form 29 August 2003
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ABSTRACT |
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glutathione ester; [3H]proline incorporation; collagen I mRNA; reactive oxygen species
Various cytokines, chemokines, and growth factors have been shown to play important roles in fibrosis development. However, transforming growth factor- (TGF-
) is considered to be the most potent and ubiquitous profibrogenic mediator (13, 24). It has been well documented that TGF-
increases the production and deposition of various ECM proteins, including collagen I, III, IV, VII, and XVI, in various types of tissues and cells (8, 14, 15, 40, 41). However, the signaling pathways or the molecular mechanisms underlying the stimulation of ECM protein production and deposition by TGF-
have not been completely elucidated. It has been reported that TGF-
decreases glutathione (GSH), the most abundant intracellular nonprotein thiol and important antioxidant in epithelial and endothelial cells (2, 31, 34, 43). Decreased GSH content has also been found in animals with experimentally induced fibrosis as well as in the lung lining fluid of patients with fibrotic lung diseases such as idiopathic pulmonary fibrosis and cystic fibrosis (3, 6, 7, 33). Most importantly, aerosolized administration of GSH or N-acetylcysteine (NAC), a precursor of GSH, attenuates experimental lung fibrosis and significantly improves pulmonary function in patients with lung fibrosis (3, 16). These observations suggest that GSH depletion plays an important role in development of fibrosis. However, the mechanism and biological significance of a decrease in GSH content in TGF-
-mediated fibrogenesis are largely unknown.
Fibroblasts play a critical role in fibrosis development, in part, through production of ECM proteins. Therefore, in this study, we examined the effects of TGF- on GSH content in murine embryo fibroblasts (NIH 3T3) and the relation between intracellular GSH content and TGF-
-stimulated collagen production. Inasmuch as GSH plays a major role in antioxidant defense, we further explored whether TGF-
stimulated production of reactive oxygen species (ROS) in NIH 3T3 cells and whether ROS were involved in the TGF-
-stimulated collagen production. Our data indicated that TGF-
decreased the intracellular GSH content in fibroblasts. Such a decrease in GSH content mediated TGF-
-stimulated collagen production. Our results also suggest that ROS are involved in TGF-
-stimulated collagen production and that GSH depletion mediates TGF-
-stimulated collagen production probably by facilitating ROS signaling.
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MATERIALS AND METHODS |
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Cell culture and treatment. NIH 3T3 cells were obtained from the American Type Culture Collection (Rockville, MD) and maintained in DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin in 5% CO2 at 37°C. The cells are spontaneously transformed mouse embryo fibroblasts and have been widely used by many investigators to study fibroblast biology. They exhibit fibroblast morphology and proliferative response to various stimuli, such as TNF-, IL-6, and TGF-
, similar to fibroblasts (14, 22, 23, 28). These cells express several types of collagens (14, 32) and collagenase (1, 42). Many signal transduction pathways, such as ERK and Rac, are intact in these cells (22, 28). Most importantly, these cells have been shown to increase their collagen synthetic rate in response to TGF-
(14). Therefore, in this study, we use this cell line as a fibroblast cell model to explore the potential role of GSH depletion in TGF-
-stimulated collagen production.
For all experiments, the cells were serum starved for 24 h (in the medium containing 0.1% fetal bovine serum) and then treated with TGF- in a serum-free medium with or without GSH modulator. Three GSH analogs, NAC, GSH, and GSH ester, were used to increase intracellular GSH content, inasmuch as they work through different mechanisms. NAC can be easily taken up by cells and hydrolyzed intracellularly to release cysteine, a rate-limiting substrate for de novo GSH synthesis (27). GSH, on the other hand, can be directly taken up by certain types of cells or can be broken down extracellularly by a membrane-bound enzyme
-glutamyltranspeptidase, providing cells with substrates for GSH synthesis (26). GSH ester is probably the most efficient way to deliver GSH to cells, inasmuch as it can be taken up by cells easily and can be hydrolyzed intracellularly to release GSH directly.
Measurement of intracellular GSH content. After treatment, the cells were washed with PBS (10 mM Na2HPO4, 1 mM KH2PO4, 137 mM NaCl, and 2.7 mM KCl, pH 7.4) three times and lysed in 5% sulfosalicylic acid. Intracellular GSH content was measured by a redox cycle assay (4). The protein content in the cells was measured by a bicinchoninic acid kit (Pierce, Rockford, IL) (36). GSH content is expressed as nanomoles per milligram of protein.
Measurement of collagen production by [3H]proline incorporation assay. Collagen production was determined by measuring incorporation of [3H]proline into collagenase-sensitive proteins, as described by Williams et al. (44). Briefly, the fibroblasts were treated with TGF- in serum-free medium containing 0.5 µCi/ml [3H]proline and 25 µg/ml ascorbic acid for indicated periods of time. After treatment, the medium was harvested and centrifuged through a Microcon-10 column and washed three times with PBS to remove free isotope. The concentrated proteins were resuspended in collagenase digestion buffer (50 mM Tris·HCl, 5 mM CaCl2, and 2.5 mM N-ethylmorpholine), half of which was used for measurement of total protein synthesized and half of which was digested with a highly purified bacterial collagenase (Sigma) for 4 h. After collagenase digestion, the proteins were precipitated using 50% TCA, and the radioactivity in the supernatant (representing collagen) was determined by liquid scintillation counting.
Northern hybridization to determine amounts of collagen I and III mRNAs. NIH 3T3 cells in 100-mm plates were treated with 1 ng/ml TGF- as described above for 6, 12, and 24 h. The cells were harvested, and RNA was isolated using TRIzol reagent according to the protocol provided by the manufacturer. Collagen I and III gene expression was assessed by Northern hybridization using collagen I- and III-specific cDNA probes, which were amplified by RT-PCR, as described by Ding and Gray (11). Twenty micrograms of RNA from each sample were resolved on a 1% agarose gel and transferred onto a nylon membrane, which was hybridized with collagen III, collagen I, and 18S cDNA, sequentially. 18S cDNA was used to normalize the amount of RNA loading. After they were washed, the membranes were scanned, and radioactivity was quantitated by an InstantImager.
Measurement of H2O2 released into the medium. H2O2 released into the medium was measured using a horseradish-linked fluorescence assay, as described previously (25). Briefly, after treatment of the cells with TGF- in serum-free DMEM, the medium was removed and the cells were treated with the same concentration of TGF-
in KRP buffer (PBS + 1.3 mM CaCl2, 1.0 mM Mg2SO4, and 10 mM HEPES, pH 7.4) containing 5 mM glucose for another 1 h. Aliquots were then withdrawn, and H2O2 concentration in the medium was determined immediately using horseradish peroxidase-catalyzed oxidation of homovanillic acid at excitation wavelength of 321 nm and emission wavelength of 421 nm. Glucose oxidase, an enzyme that generates H2O2 continuously, was used as a positive control.
Determination of superoxide production. Superoxide production in the cells was measured by a lucigenin-coupled chemiluminescence assay, as described previously by Xie et al. (45). Briefly, the cells were washed with PBS after treatment, resuspended in Tris-sucrose buffer (pH 7.4), and lysed by a freeze-thaw process. Measurement of superoxide production was started by addition of lucigenin (final concentration 5 µM) and NADH (100 µM) to the reaction tube. Generation of luminescence was monitored continuously for 10 min using a Sirius single-tube luminometer (Zylux). To minimize lucigenin's amplification of ROS formation, a low concentration of lucigenin (5 µM) was used.
Statistics. Values are means ± SE. Data were evaluated by oneway ANOVA. Statistical significance was determined by Fisher's least significant difference test. P < 0.05 was considered significant.
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RESULTS |
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TGF- increased the steady-state level of collagen I mRNA. Collagen I and III are the two major types of fibrillar collagens that have been shown to be induced by TGF-
in various types of tissues or cells. To further explore the mechanism whereby TGF-
increases collagen protein production, we examined the effect of TGF-
on the steady-state levels of collagen I and III mRNAs by Northern analysis. The increase in collagen protein production was associated with an increase in the steady-state level of collagen I, but not collagen III, mRNA (Fig. 2). Importantly, the increase in collagen I mRNA also occurred after GSH was decreased (6 h), which further suggests that GSH depletion may have a causative effect on TGF-
-stimulated collagen production.
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Modification of intracellular GSH content altered TGF--stimulated collagen production. To determine whether GSH depletion caused by TGF-
plays a role in TGF-
-induced collagen production, we studied the effects of NAC (Fig. 3A), GSH (Fig. 3B), and GSH ester (Fig. 3C) on TGF-
-induced collagen production. These GSH analogs restored the intracellular GSH content in TGF-
-treated cells to the control level (Fig. 3, right) and partially (in the case of NAC) or completely (in the cases of GSH and GSH ester) blocked TGF-
-stimulated collagen production (Fig. 3, left). GSH, at high concentration, and GSH ester not only blocked TGF-
-stimulated, but also reduced the basal level of, collagen production.
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In further support for a role of GSH depletion in TGF--stimulated collagen production, we pretreated the fibroblasts with BSO to inhibit de novo GSH synthesis. BSO decreased the intracellular GSH content (Fig. 4, right) and enhanced TGF-
-stimulated collagen production (Fig. 4, left). Correlation analysis using the above data (Figs. 3 and 4) showed that the intracellular GSH concentration was inversely correlated with the rate of [3H]proline incorporation in TGF-
-treated cells (r = 0.803, P < 0.01).
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TGF- increased ROS production in NIH 3T3 cells. Because GSH plays an important role in antioxidant defense and TGF-
increases ROS production in other experimental systems, we examined whether TGF-
also stimulated ROS production in NIH 3T3 cells and whether GSH has an effect on this process. The results showed that H2O2 release was significantly increased after the cells were treated with TGF-
for 8 h (0.27 ± 0.08 in TGF-
-treated vs. 0.09 ± 0.08 µM in control cells). H2O2 concentration in the medium was also significantly increased when the cells were treated with glucose oxidase (2.7 ± 0.2 in glucose oxidase-treated vs. 0.09 ± 0.08 µM in untreated control cells). Pretreatment of the cells with GSH ester reduced H2O2 accumulation in the medium of cells treated with TGF-
+ glucose oxidase (Fig. 5A), suggesting that GSH depletion may facilitate accumulation of H2O2 in TGF-
-treated cells.
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The potential source of H2O2 was further investigated by measuring superoxide production after TGF- treatment in the presence or absence of DPI, an NAD(P)H oxidase inhibitor. Superoxide production was significantly increased 4 h after TGF-
treatment and peaked at 16 h. DPI reduced the basal level of superoxide production and completely blocked TGF-
-stimulated superoxide production (Fig. 5B). These data suggest that increased activity of NAD(P)H oxidase may contribute to increased ROS production in TGF-
-treated NIH 3T3 cells. The results also suggest that ROS production is independent of GSH depletion in TGF-
-treated cells as GSH was depleted 6 h after TGF-
treatment.
ROS were involved in TGF--stimulated collagen production. To clarify whether ROS plays a role in TGF-
-stimulated collagen production, we examined the effect of H2O2, added directly to the medium or generated continuously in the medium by exogenously added glucose oxidase, on TGF-
-stimulated collagen production. The results showed that although neither H2O2 nor glucose oxidase alone at the concentration used had a significant effect on collagen production, they significantly enhanced TGF-
-stimulated collagen production. H2O2 at 20 µM enhanced TGF-
-stimulated collagen production by 52%, whereas 0.05 mU/ml glucose oxidase caused 37% enhancement and 0.2 mU/ml glucose oxidase caused 56% enhancement (Fig. 6A).
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To further confirm the role of ROS in TGF--stimulated collagen production, we inhibited ROS production by pretreating the cells with 2 µM DPI for 1 h and then with TGF-
in the absence of DPI for another 24 h. The results showed that inhibition of ROS production significantly reduced the basal level of collagen production and completely blocked the TGF-
-stimulated collagen production (Fig. 6B). The results further indicated that ROS might serve as a signaling molecule to regulate collagen production under basal and TGF-
-stimulated conditions.
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DISCUSSION |
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TGF- signaling pathway, from receptors to nucleus through Smad proteins, which mediates the induction of many TGF-
-responsible genes, has been described in detail elsewhere (10, 12, 18). However, the signaling pathways that specifically mediate TGF-
-stimulated ECM protein production have not been well elucidated. Interestingly, it has been reported that TGF-
stimulates ROS production while ROS mediate various TGF-
effects, such as growth inhibition/apoptosis, production of IL-6 and macrophage colony-stimulating factor, and expression of early growth response-1 (9, 17, 19, 21, 29). Whether ROS is also involved in TGF-
-stimulated collagen production has been less well studied. In this study, we showed that TGF-
increased superoxide production and H2O2 release in fibroblasts (Fig. 5). Increasing ROS level by directly adding H2O2 to the medium or by treating the cells with glucose oxidase enhanced TGF-
-stimulated collagen production. Most importantly, suppression of superoxide production by inhibiting the activity of NAD(P)H oxidase, the enzyme responsible for the increased ROS production in TGF-
-treated cells (37, 38), dramatically reduced basal and completely blocked TGF-
-stimulated collagen production (Fig. 6). These data suggest that ROS may be generated by NAD(P)H oxidase in NIH 3T3 cells in response to TGF-
and that ROS are important not only for TGF-
-stimulated but also for basal collagen production. Although it has been well documented that TGF-
stimulates ROS production, where in the cell ROS are generated is unclear. Some results showed that ROS were generated on the cell surface by NADH oxidase, whereas others suggested that they might come from intracellular sources (21, 29, 37, 38). The methodology used in this study cannot distinguish an intracellular from an extracellular source for ROS. However, inasmuch as H2O2 can diffuse freely into and out of cells, H2O2 generated or added to the outside of cells will still be able to exert biological or pathological effects intracellularly. Although H2O2 enhanced TGF-
-stimulated collagen production, increasing ROS level alone (treatment of the cells with H2O2 or glucose oxidase alone) failed to stimulate collagen production (Fig. 6A). These data suggest that ROS is essential, but not sufficient, for collagen production and that one or more factors in addition to ROS are also involved.
GSH, the most abundant intracellular free thiol, participates in many important biological processes, such as metabolism of endogenous and exogenous compounds, cell proliferation, and regulation of gene expression. However, the best-described function of this tripeptide is detoxification of oxidants by directly scavenging free radicals or acting as a coenzyme in glutathione peroxidase-catalyzed reactions. When GSH content decreases, the capacity of the cells to remove ROS through glutathione peroxidase is diminished, and therefore ROS accumulate. One potential mechanism underlying the effect of GSH depletion on TGF--stimulated collagen production is that GSH depletion extended TGF-
-initiated ROS signaling processes as a result of decreased capacity of the cells to remove ROS. This conclusion is drawn on the basis of the fact that TGF-
decreased the intracellular GSH content by 6 h. However, H2O2 accumulation in the medium was not detected until 8 h after TGF-
treatment, even though superoxide production was increased by 4 h. Increasing intracellular GSH content decreased basal as well as TGF-
+ glucose oxidase-stimulated H2O2 accumulation. These data suggest that GSH plays an important role in removing ROS in NIH 3T3 cells, and GSH depletion may facilitate ROS accumulation. Similar results have also been observed in TGF-
-treated human lung fibroblasts, in which an increase in H2O2 release was observed after the cells were exposed to TGF-
for 8 h and peaked at 16 h (21, 38). Therefore, GSH depletion mediated TGF-
-stimulated collagen production, probably by facilitating ROS accumulation. However, decreasing GSH per se, such as in cells treated with BSO alone, is not sufficient to stimulate collagen production, although it enhanced TGF-
-stimulated collagen production (Fig. 4). These data further suggest that GSH depletion/ROS production is essential but not sufficient for collagen production, and one or more other factors are also involved. A schematic diagram of the relationship between GSH depletion, ROS, and TGF-
-stimulated collagen production is presented in Fig. 7.
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Although turnover of collagen is generally considered slower than that of most other proteins, a large body of evidence has shown that turnover of some collagens, especially the newly synthesized collagens, is very fast and that collagen synthesis and degradation start almost simultaneously. Therefore, an increase in [3H]proline incorporation during a 24-h incubation period, as shown in this study, could result from an increased collagen synthesis and/or a decreased collagen degradation. In this study, we showed that TGF- increased collagen I mRNA, suggesting that the increased [3H]proline incorporation results, at least in part, from an increased collagen synthesis. It has also been well documented that TGF-
increases the activities of various proteinase inhibitors, such as tissue inhibitors of metalloproteinases and plasminogen activator inhibitors, and decreases the activities of various metalloproteinases, leading to decreased ECM degradation and fi-brosis (30, 35, 39). Whether decreased collagen degradation also contributes to TGF-
-induced collagen production in NIH 3T3 cells remains to be examined. Most importantly, which step(s), collagen synthesis and/or collagen degradation, is affected by GSH depletion needs to be further explored.
The suppression of GSH concentration with TGF- that we observed in fibroblasts is consistent with the earlier findings of other investigators in different types of cells (2, 5, 20). The potential mechanisms for a decrease in intracellular GSH content include 1) increased consumption, due to exposure to massive oxidants or decreased GSH reductase activity, which leads to a reduction of redox cycling of GSSG and, therefore, an increased loss of GSH in the GSSG form, 2) decreased GSH synthesis, due to decreased availability of substrates and/or the activities of the enzymes involved in this process, and/or 3) increased GSH efflux or transport. The mechanism underlying the decrease in GSH content in TGF-
-treated fibroblasts is not clear. It has been proposed that decreased synthesis as a result of a decline in the uptake of GSH precursor amino acid (5) or a downregulation of the gene expression of glutamate cysteine ligase, the rate-limiting enzyme in de novo GSH synthesis, was responsible for TGF-
-induced GSH depletion in epithelial and endothelial cells, respectively (2, 20). Whether the same mechanism, increased consumption, or increased efflux/transport underlies the GSH depletion in TGF-
-treated fibroblasts remains to be explored. Further studies are warranted as revealing the mechanism underlying the TGF-
-induced decline in GSH content in fibroblasts will provide important information not only for understanding the mechanism of TGF-
fibrogenesis but also for developing effective therapeutic agents to prevent and treat these fibrotic diseases.
Finally, we address some limitations of this study. 1) Although NIH 3T3 cells have been widely used to study fibroblast biology, they are derived from mouse embryo. Thus extrapolation of the observations to human diseases may be difficult. Future study will focus on primary human lung fibroblasts. 2) DPI may have some nonspecific effect, even under our mild treatment condition. Therefore, interpretation of the results may not be straightforward. 3) Although correlation study shows that intracellular GSH contents are inversely correlated with the rates of [3H]proline incorporation in TGF--treated cells, the GSH modulators might have some extracellular effects in addition to increasing intracellular GSH content. Further study will employ different strategies to modulate specifically intracellular GSH, for example, by genetically modifying the expression of glutamate cysteine ligase, the rate-limiting enzyme in de novo GSH synthesis, to further confirm our finding that intracellular GSH level affects TGF-
-stimulated collagen production.
In summary, the data presented here showed that TGF- decreased GSH content in fibroblasts and the decrease in the intracellular GSH content mediated TGF-
-stimulated collagen synthesis, perhaps by facilitating ROS signaling processes. These findings provide new insights into the mechanism of TGF-
fibrogenesis and may suggest new strategies for treatment of fibrotic diseases.
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ACKNOWLEDGMENTS |
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GRANTS
The work was supported by National Institutes of Health Grants ES-011831 (to R.-M. Liu) and HL-58655 (to M. Olman) as well as a Young Investigator Award (to R.-M. Liu) from the University of Alabama at Birmingham Center for Free Radical Biology and a Veterans Administration Merit Award (to M. Olman).
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FOOTNOTES |
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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.
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REFERENCES |
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