Previous research regarding CS-induced oxidative stress has focused on irreversible oxidations linked to damage, while ignoring the effects of CS on physiologically relevant oxidations that can reversibly modify function. The objective of the present study, therefore, was to investigate whether CS can cause changes in protein S-glutathionylation, an oxidation that can be reversed by Grxs, and if changes in this S-glutathionylation–Grx1 redox system play a role in epithelial cell death provoked by CS.
This is the first report to demonstrate attenuation of Grx1 expression and Grx activity by CSE, in concert with increased protein S-glutathionylation in lung epithelial cells. In patients with COPD, it has been shown that the number of Grx1-positive macrophages was decreased in the lungs, along with decreases in Grx1 protein levels in whole-lung homogenates. In contrast, in sputum supernatants, more Grx1 was detected during acute exacerbations (13
). Protein S-glutathionylation was not investigated in the latter study, but elevated levels were reported in blood samples of smokers compared with nonsmokers (22
). The present study in cell culture models confirms results of the previous reports regarding the modulation of Grx1 expression and protein S-glutathionylation in patients with COPD and healthy smokers. However, CS probably did not directly affect mRNA expression of Grx1, as attenuated levels of Grx1 mRNA could only be observed after at least 24 hours of exposure. It would appear more likely that CS acts on signaling pathways that modulate transcription factors that, in turn, regulate Grx1 mRNA expression. Interestingly, the decreased expression of Grx1 observed after transforming growth factor (TGF)–β treatment also occurred only after 24 hours (mRNA ) or 72 hours (protein [23
]). The signaling intermediates and transcription factors involved in the modulation of Grx1 mRNA remain to be investigated. There is only a single study regarding potentially important transcription factor binding sites and regulatory regions in the human Grx1 promoter (24
), both of which need to be evaluated in detail in future research. CSE and TGF-β both down-regulate Grx1 mRNA expression, which, for TGF-β, appears to fit into a general repressive effect on antioxidant genes, whereas this is not the case for smoke. No effects of CS on Grx2 mRNA were observed, which is in line with previous studies in which only levels of Grx1, but not of Grx2, were affected (12
). It is, however, possible that the activity of Grx2 is altered by smoke exposure, as this isoform is activated when the active site is opened up upon monomerization, which can be accomplished by oxidation (25
). Furthermore, the activity assay used here does not distinguish between the different isoforms. Together, these observations could explain why the strong effects observed on Grx1 expression and on recombinant Grx1 activity after CSE exposure do not translate into equally strong effects on total cellular Grx activity.
In addition to the attenuated expression of Grx1 in response to CSE exposure, we observed elevated levels of Grx1 mRNA, protein, and activity in control cells over time in culture. Accordingly, protein S-glutathionylation levels were also decreased over time in culture. Some previous reports have linked Grx1 to cell proliferation. For instance, the enzyme was first discovered as an alternative electron donor for ribonucleotide reductase in Escherichia coli
, an enzyme essential to DNA synthesis in proliferating cells (26
). In addition, Grx1 has been shown to control actin S-glutathionylation and its polymerization status after growth factor stimulation, which was postulated to play a role in the formation of signal transduction scaffolds and the cellular response to growth factors (27
). The increased levels of Grx1 in culture over time could potentially be linked to proliferation, as the experiments were performed at subconfluency, and minor proliferation could still be observed using 0.5% FBS.
In the present study, we show that CS not only attenuated Grx1 expression, but that the Grx1 protein itself was modified by CSE, thereby decreasing its activity ( and ). It was determined that CSE exposure resulted in Grx1 adduct formation through both alkylation by acrolein and carbonylation. Acrolein is the most highly oxidative compound in CS, and is known to irreversibly bind proteins, probing them for rapid proteolytic degradation (6
). It is therefore plausible that alkylation of Grx1 leads to proteolytic degradation, a scenario that needs to be formally tested. Nonetheless, results from the present study demonstrate that CS targets Grx1 via multiple mechanisms, which has implications for cell survival.
Cysteines with a low acid dissociation constant are prone to S-glutathionylation upon mild oxidative stress, and when S-glutathionylation occurs at a critical cysteine, this can modify the activity and conformation of the targeted protein. In the present study, we demonstrate that CS exposure enhanced total levels of protein S-glutathionylation (). Further studies are needed to investigate which particular proteins are targeted by S-glutathionylation. The function of proteins potentially involved in disease pathogenesis, such as inhibitory κB kinase β and NF-κB, activator protein 1, and matrix metalloproteases, have been shown to be affected by S-glutathionylation and, in some instances, by alterations in Grx1 levels (9
). Variations in Grx1 and S-glutathionylation of these proteins could, therefore, contribute to the pathophysiology of COPD.
Some of the target proteins of S-glutathionylation are known to modulate cell death (18
), a process that has raised interest as a mechanism in the development of COPD (29
). Here, we demonstrate that modulation of Grx1 expression, in conjunction with alterations in protein S-glutathionylation, in lung epithelial cells affects their survival in response to CS. So far, Grx1 has been reported to have a cardioprotective role and reduce ROS production after ischemia and reperfusion in Glrx1
transgenic mouse hearts. Conversely, Glrx1−/−
mice and Grx1 inhibition by cadmium increased infarct size and ROS production (30
). In addition, lens epithelial cells of Glrx1−/−
mice exhibited increased sensitivity to oxidative stress, as they had a reduced ability to clear H2
, and administration of recombinant Grx1 restored antioxidant capacity (31
). In the present study, we show that primary MTE cells isolated from Glrx1−/−
mice were more sensitive to CS-induced cell death compared with wild-type control animals, in association with enhanced protein S-glutathionylation. Conversely, overexpression of Grx1 in an epithelial cell line was found to protect against CS-induced cell death, while attenuating the induction of S-glutathionylation in response to CSE. Collectively, these data indicate that the decreased expression of Grx1 and attenuation of Grx activity after CSE exposure are indeed responsible for observed increases in total protein S-glutathionylation, and contribute to CSE-induced death of lung epithelial cells. However, additional studies need to be conducted to unravel the target proteins for increased S-glutathionylation that contribute to cell death after CS exposure. Mediators of apoptosis and cell death shown to be modulated by the S-glutathionylation/Grx1 axis include procaspase-3 (32
), multiple members of the NF-κB survival pathway (33
), ASK1 (34
), and Fas (18
Taken together, the data show increasing evidence for Grx1 as a potential therapeutically relevant candidate for enhancing cell survival upon CS exposure. A previous study showed a similar protective effect using recombinant thioredoxin-1, another member of the thioredoxin family, in the CS exposure model for COPD in mice (35
). Restoring Grx1 content in the lungs after exposure to CS may, therefore, have implications in enhancing cell survival, and thus potentially help to prevent the development of emphysema.