Eaton and colleagues (79
) analyzed the effects of cardiac ischemia–reperfusion on glutathionylation of the cardiac proteome. Isolated rat hearts were perfused with biotinylated GSH following stop-flow ischemia, and glutathionylated proteins were detected via
blotting with streptavidin-HRP. According to this analysis, overall protein glutathionylation was increased ~15-fold following ischemia–reperfusion (IR), with the majority of the glutathionylation events occurring early in the reperfusion period. Some caution is required in interpreting these results because trapping the modified proteins as protein–SSG-biotin may inhibit their deglutathionylation and overestimate the degree of glutathionylation that would occur otherwise (273
In the same study, glyceraldehydes-3-phosphate dehydrogenase (GAPDH) was identified as a prominent cardiac protein glutathionylated during IR. GAPDH immunopurified from ischemic tissue exhibited DTT-reversible loss of function, suggesting that GAPDH glutathionylation is likely inhibitory in vivo.
While the consequences of GAPDH inhibition on cardiac function were not explored in this study, logical possibilities include: (a) contribution to the blockade of glycolysis characteristic of ischemic injury, (b) interference with translocation to the nucleus, resulting in increased apoptosis (51
), or (c) little to no effect, with the modified cysteine of GAPDH, serving primarily as a “sink” for excess oxidants rather than a site of homeostatic regulation. Importantly, GAPDH activity was restored by the end of the reperfusion period, suggesting that for this protein at least, glutathionylation may serve as a temporary modification to protect catalytic cysteines from irreversible oxidation.
Evidence for actin glutathionylation was demonstrated in a rat model of in vivo
). Homogenates of ischemic tissue subjected to Western blot analysis with an anti-GSH antibody exhibited a prominent band corresponding to the molecular weight of actin, and immunoprecipitated actin reacted with the same antibody in a DTT-reversible manner. Studies on isolated G-actin indicated that glutathionylation delayed its rate of polymerization [consistent with a previous study on the effect of actin glutathionylation in A431 cells (315
)], and decreased the cooperativity of its binding to tropomyosin, suggesting that actin-SSG formation contributes to the decline in cardiac contractility observed during ischemia. This interpretation could be strengthened by determining the glutathionylation status of actin following reperfusion. Since contractility is generally recovered in this model by the end of the reperfusion period, it would be expected that actin-SSG levels would decline with a similar time course, providing the basis for the improved contractility following IR insult.
In contrast to actin and GAPDH, mitochondrial complex II exhibits the opposite glutathionylation pattern following IR (i.e.
, deglutathionylation). In vivo
IR, as well as stop-flow ischemia of isolated rat heart, resulted in decreased immunoreactivity of the 70 kD subunit of complex II (i.e.
, SQR) with an anti-GSH antibody (39
). Studies of isolated SQR indicated that glutathionylation increased electron transfer activity somewhat and decreased leakage of superoxide (O2·−
), suggesting that IR-associated deglutathionylation
contributes to the decrease in SQR function observed during IR.
What could explain the divergence between the glutathionylation pattern of the general cardiac proteome (including actin and GAPDH) and that of SQR during IR? Possible contributing factors include the accessibility and intrinsic reactivities of modified cysteines, their proximity to sites of ROS production (as well as to Grx), and structural features that may stabilize the glutathionylated—or thiolate—status of the modified cysteine. Overall, the divergent results of these studies reinforce the concept that a single oxidative stimulus (e.g., IR) can affect glutathionylation status of different protein cysteines in different directions. Moreover, understanding the basis for these differences will require a greater understanding of the regulation of the factors regulating protein glutathionylation (e.g., concentrations of ROS, Grx1, and Grx2) within specific intracellular compartments, as well as quantitative relationships among protein–SSG events, the magnitude of alteration in protein activity, and resulting impact on cellular function.
Understanding the role of protein glutathionylation in myocardial infarction can also be approached by manipulating Grx, the primary intracellular deglutathionylating enzyme (41
). To this end, mouse models with embryonic knockout of Grx1, as well as overexpression of Grx1 and Grx2 transgenes, were developed and subjected to in vivo
and ex vivo
IR. In general, experiments with transgenic animals suggest a cardioprotective role for both Grx isoforms; however, additional studies are needed to link the effects of each transgene to the protein glutathionylation status.
The first group to investigate the role of Grx on IR injury tested the effect of Grx1 embryonic knockout on infarct size and area at risk in an in vivo
model of IR (120
). No difference in either parameter was observed in Grx1 knockout (KO) vs.
WT animals, even though the deglutathionylase activity of all of the mouse tissues, including heart, was essentially absent. One possible explanation for this unexpected outcome is that compensatory changes in the mechanisms of cellular homeostasis occurred during development, offsetting the detrimental effect of Grx1 knock-out. Therefore, to circumvent this complication, we recommend that future studies on the effects of Grx1 in IR injury utilize a tissue-specific, inducible KO model.
Malik et al.
) further explored the role of Grx1 in IR injury by comparing the effects of embryonic global glrx1
KO with muscle-specific overexpression, and by widening the scope of injury to include ischemic pre-conditioning (IPC) prior to IR. IPC is widely recognized to decrease subsequent IR injury (reviewed in refs. 75
), and there is evidence that Grx1 contributes to regulation of several signaling pathways implicated in the mechanism of IPC (see below). Unlike Ho et al.
), Malik and colleagues reported a small but significant increase in infarct size, as well as decreased contractile performance, in glrx1
KO mice compared to controls. In contrast to the effects of Grx1 KO, Grx1 overexpression appeared to decrease infarct size and protect coronary function.
The basis for the distinct effects of glrx1
KO on infarct size reported by these two groups is not obvious, but likely reflects differences in experimental protocols. For example, Grx1 might play an important role in cardioprotection during early reperfusion, thus affecting infarct size after 2
h of reperfusion [observed by Malik et al.
)] but exhibiting less of an effect after 4
h [when Ho et al.
) measured infarct size]. Alternatively (or additionally), neurohumoral factors might have blunted the effect of glrx1
KO in the in vivo
IR model of Ho et al.
)., while their absence revealed an important cardioprotective role for Grx1 in the isolated heart model of Malik et al.
). This scenario predicts that glrx1
KO indeed confers a detrimental effect on cardiac function during IR, and compensation by other organ systems may represent an important component of disease outcome when Grx1 activity is perturbed.
More dramatic than the role of Grx1 in IR injury was its contribution to IPC. Grx1 overexpression potentiated the protective effect of IPC on infarct size and cardiomyocyte apoptosis, while IPC failed to confer any cardiac protection in Grx1 KO mice. To explore the mechanism of Grx1-associated protection vs.
IR injury, ROS production (via
malondialdehyde (MDA) content), Akt phosphorylation, and Bcl-2 content were assayed. While there was no apparent change in Bcl-2 content associated with change in Grx1 content, Grx1 expression inversely correlated with MDA content following IR, and Grx1 KO was associated with decreased IPC-induced Akt phosphorylation. The latter observation led the authors to suggest that Grx1 could contribute to IPC via
activation of Akt, which is consistent with other reports linking Grx activity to Akt phosphorylation (207
), although the mechanism for this effect in vivo
is not yet established (see Preconditioning section, below). However, the additional conclusion that Grx1's cardioprotective effects can be attributed to its role as an “antioxidant” is problematic. First, it is not consistent with the primary (and well-documented) function of Grx as a deglutathionylating enzyme (41
). In fact, studies of protein glutathionylation were not reported here, so it cannot be concluded that Grx1's cardioprotective effects were not related to its role in protein deglutathionylation. Second, a Grx1-mediated antioxidant activity is not explicitly demonstrated. One could imagine that, if Grx1 were expressed at high enough levels, it could serve as an antioxidant by supplying reduced cysteines in a manner analogous to N
-acetylcysteine or DTT, but it is unlikely that such concentrations could be reproduced in vivo
, even within a disease context. Is there a more likely explanation for the decreased MDA content observed in Grx1 TG mice subjected to IR? One possibility is that Grx1 may indirectly decrease ROS by deglutathionylating a ROS-generating enzyme, such as complex I, which exhibits increased superoxide production upon glutathionylation (293
). This interpretation is more consistent with documented activities of Grx1, and could be investigated by determining the glutathionylation status of complex I during IR in WT and Grx1 TG mice. A similar scenario has been described for the cardioprotective role of Trx1 function in ischemic preconditioning. Analogous to glrx1
KO, inhibition of Trx1 resulted in increased MDA formation in the IR heart, suggesting an “antioxidant” function for the enzyme (62
). Instead of proposing that Trx itself serves as an antioxidant, the authors suggested that it might promote antioxidant activity via
up-regulation of antioxidant enzymes such as MnSOD. In light of their distinct enzymatic activities, it is intriguing that Grx1 and Trx1 confer similar cardioprotective effects during IPC, and determining the mechanism(s) for each enzyme's protective effects represents a fascinating avenue for future study.
The role of Grx2 in IR injury was also investigated using a transgenic animal model (211
). As with Grx1 overexpression, Grx2 overexpressing mice exhibited decreased infarct size and improved myocardial function following ex vivo
IR compared to WT mice. Unlike Grx1, Grx2 appeared to influence apoptosis, with Grx2 TG mice showing fewer apoptotic cardiomyocytes following IR compared to control animals. Additional evidence supporting a cardioprotective effect of Grx2 included decreases in caspase activation, cardiolipin loss, MDA formation, and diminution of the GSH:GSSG ratio following IR. The roles of Grx2 in cytochrome c release and Akt phosphorylation were less straightforward. Grx2 TG animals exhibited decreased IR-induced cytochrome c release, but the amount of cytosolic cytochrome c was much higher at baseline in TG animals and appeared to decline
with IR. Grx2 overexpression has been linked to decreased cytochrome c release in oxidant-challenged HeLa cells (85
), but the basis for its loss from the cytosol in Grx2 TG hearts with IR is puzzling. Phosphorylated Akt was also higher at baseline in TG vs.
WT animals, with levels remaining steady following IR. Considering the distinct subcellular localizations of Grx2 and Akt [Grx2 in the mitochondrial matrix (226
) and Akt in the cytosol], this regulation is most likely indirect; however, a direct role of Grx2 in regulating Akt activity [as has been proposed for Grx1 (207
)] is possible if the Grx2 transgene was also expressed in cytosol. Since cytosolic Grx activity was not reported in the study, this explanation cannot be ignored. In fact, Enoksson and co-workers (85
) showed that Grx2 targeted to the cytoplasm protected HeLa cells from doxorubicin-induced apoptosis, so verification of appropriate subcellular localization is a critical prerequisite for interpreting the findings of any study in which Grx2 is overexpressed.
Overall, the work of Nagy et al.
) suggests a role for Grx2 in cardioprotection, but as in the case of other studies documenting the cytoprotective effect of Grx2 (85
), mechanism(s) remain unknown. Importantly, candidate target pathways were proposed, including Akt and NF-κB. Future work should focus on identifying the direct targets of Grx2 responsible for its pleiotropic effects on oxidant-induced signaling. An attractive candidate is mitochondrial complex I, in which glutathionylation of the 51- and 75-kD subunits is correlated with electron transport inhibition and increased production of superoxide (293
). The 51kD subunit contains one conserved cysteine that is not bound to an FeS cluster, and this cysteine faces the mitochondrial matrix (119
), making it a potential site of regulation by Grx2. Glutathionylation of complex I, with associated increases in superoxide production, would be expected to increase cytochrome c release and caspase activation, induce survival signals, and contribute to infarct size and cardiac dysfunction. Thus, complex I deglutathionylation by Grx2 is a conceivable upstream event responsible for modulating these effects in Grx2 transgenic animals.
Finally, the roles of both Grx isoforms in IR injury were explored in rats by Mukherjee et al.
). These investigators examined the effects of broccoli extract (administered by oral gavage) on IR injury in isolated working rat hearts. Rats fed normal diets exhibited decreased Grx expression during IR, while rats given broccoli extract showed preservation of Grx1 and Grx2 content, which correlated with decreases in infarct size, cardiomyocyte apoptosis, cytochrome c release, and caspase-3 activation, as well as improved post-MI cardiac and hemodynamic function. It is difficult, however, to determine the specific contribution of Grx isoforms to these cardioprotective effects, since broccoli gavage also induced other genes known to regulate cellular survival and redox homeostasis (e.g.
, thioredoxins 1 and 2, thioredoxin reductase), and the effect of broccoli gavage on protein glutathionylation status was not reported.
In summary, cardiac ischemia-reperfusion results primarily in increased protein glutathionylation; however, some proteins (e.g., complex II) are deglutathionylated during an IR episode. The effects of glutathionylation on function of these proteins (e.g., GAPDH, actin) may protect them from irreversible damage, or contribute to IR-induced injury. The latter possibility is supported by the cardioprotective phenotype of Grx TG animals, which exhibit decreased infarct size and improved cardiac function following IR. While data from KO animals are not as straightforward, studies on mice with inducible, tissue-specific KO might clarify the roles of Grx in acute IR injury. A critical frontier in this endeavor is to determine the mechanism(s) of Grx-related cardioprotection, which will require linking the protective effects of Grx on cardiac function with glutathionylation status of its molecular targets.
Nonspecific oxidative injury
IR and IPC are both conditions of oxidative stress. In IR, ROS are produced primarily from complexes I and III of the mitochondrial electron transport chain (167
), and ROS generation from mitochondria and/or NADPH oxidase appears to contribute to PC signaling (75
). Above, we discussed evidence for increased glutathionylation of the intracellular proteome—as well as individual proteins—with IR, and the potential for regulation of survival proteins by glutathionylation during IPC. Importantly, additional proteins are reported to be regulated by glutathionylation during generalized oxidative challenges, such as H2
treatment or exposure to decreased GSH:GSSG ratios. Although these oxidative stimuli do not necessarily model the physiological state or a specific disease condition, they identify candidate proteins for regulation by glutathionylation during pathological oxidative stresses such as IR, as well as other cardiovascular diseases associated with oxidative stress, such as hypertension (113
) and atherosclerosis (151
For example, mitochondrial complex I is glutathionylated in vitro
upon exposure to GSSG (293
) or low GSH:GSSG ratios [i.e.
, 0.67–12, (23
)], and glutathionylation is reversed upon incubation with Grx2 and GSH. Complex I glutathionylation results in increased superoxide production (293
), suggesting that this modification would increase ROS generation, leading to activation of redox signaling pathways and/or induction of cell death, depending upon the magnitude of modification. Key considerations regarding the possibility of complex I regulation by glutathionylation in vivo
include its mechanism of formation and its potential role in cardiac disease.
Taylor et al.
) propose that oxidative stress within the mitochondria alters the GSH:GSSG ratio sufficiently to cause complex I–SSG formation by thiol disulfide exchange; however, this mechanism is unlikely unless the modified cysteines display unusually low redox potentials (92
). Thus, alternative mechanisms of glutathionylation (e.g., via
nitrosothiol intermediate, sulfenic acid intermediate, etc.
, as described above) are more probable (). An additional mechanism of glutathionylation was proposed by Beer et al.
), namely, catalysis by Grx2. Although mammalian glutaredoxins are efficient protein deglutathionylating
enzymes, Grx1 promotes protein glutathionylation
in the presence of GS·
) and—to a much lesser extent—GSNO or GSSG. To determine if Grx2 exhibited similar behavior, it was incubated with 5
GSSG and mitochondrial membranes from rat heart containing complex I. Addition of Grx2 accelerated glutathionylation of membrane thiols over a short time course; however, when GSH was added to glutathionylated membrane proteins, Grx2 incubation led to overall deglutathionylation
of protein–SSG. Since the latter conditions more closely represent the intermitochondrial milieu, they better reflect the potential environment in which Grx2 may regulate complex I–SSG in vivo.
Thus, catalysis of glutathionylation by Grx2 appears not to be a likely mechanism of complex I–SSG formation.
Whether complex I is indeed regulated by glutathionylation in the intact heart has not yet been explored. Studies focused on documenting complex I–SSG formation in cardiac cells or tissue, with an oxidative stimulus relevant to cardiac disease (e.g., IR, angiotensin II treatment), and attention to the effects of Grx1 and Grx2 on complex I glutathionylation status will provide additional insight into this potential contribution to cardiac injury.
Another mitochondrial enzyme potentially regulated by glutathionylation during cardiac oxidative stress is α-ketoglutarate dehydrogenase (KGDH). Nulton–Persson and colleagues (217
) demonstrated that H2
treatment of rat heart mitochondria led to inhibition of KGDH activity, which was reversed by Grx1 and GSH within minutes, but unaffected by the Trx system. Although KGDH glutathionylation was not shown directly, it was inferred from the recovery of activity by Grx1 treatment, and hypothesized to protect catalytic cysteine residues from irreversible damage during oxidative stress conditions, such as IR.
A later study by the same group (8
) focused on the glutathionylation site of KGDH, and proposed an intriguing model in which glutathionylation occurs on a covalently bound lipoic acid moiety, rather than the typical cysteine sulfhydryl moiety. This conclusion was based on observations that H2
treatment prevented recognition of KGDH by an antilipoate antibody, as well as HNE-mediated oxidation. A key question concerning the proposal of mixed disulfide formation between KGDH-lipoic acid and GSH is its mechanism of stabilization. It would be expected that the vicinal cysteine on lipoic acid would undergo thiol–disulfide exchange with its neighboring Cys-SSG, forming lipoic acid intramolecular disulfide and GSH. Structural analysis or modeling studies might identify potential residues that stabilize the second, reduced Cys on lipoic acid, making it unavailable to react with the neighboring Cys-SSG. While stable mixed disulfide formation between protein-bound lipoic acid and GSH is a novel concept, and catalysis of lipoic acid-SSG would represent a new activity for Grx1, there are alternative interpretations to the authors' observations. For example, it is possible that KGDH is glutathionylated on a cysteine residue in close proximity to the bound lipoic acid, and steric interference by this glutathionylated cysteine blocks accessibility of lipoic acid to antibodies and HNE. Alternatively, glutathionylation on a distant Cys could induce a conformational change with the same effect on access of the lipoic acid to detection reagents. This possibility could be addressed by analyzing the glutathionylated product by mass spectrometry, and/or by isolating the lipoyl moiety prior to analysis for S
Overall, glutathionylation of complex I and KGDH appear to be facile upon exposure of mitochondria to oxidants in vitro; however, their relevance to oxidative stress-associated cardiac disease is not yet established. Determination of their glutathionylation status with IR, or other pathophysiological oxidative challenge, will provide insight into the likelihood of their regulation by glutathionylation in vivo.
Atherosclerosis is a complex disease process involving interactions between multiple cell types in the blood and vasculature. The precise role of glutathionylation in the development and progression of atherosclerosis is unknown; however, conditions within atherosclerotic plaques (e.g.
, hypoxia, oxidative stress, oxLDL, and inflammation) have been shown in other contexts to promote glutathionylation (79
), and Grx has been reported to associate with areas of oxidative stress within the vasculature (221
). The following discussion explores further evidence for involvement of protein glutathionylation in atherogenesis.
Global protein glutathionylation increases in human monocyte-derived macrophages exposed to oxidized LDL (oxLDL) (319
), a major component of atherosclerotic plaques also believed to contribute to their progression (151
). Together with GSH depletion, increased protein–SSG content was implicated in oxLDL-induced macrophage death in vitro.
Dying macrophages represent a major component of atherosclerotic plaques, and their presence in atherosclerotic lesions increases risk of rupture (164
). The role(s) of specific glutathionylated proteins in macrophage cell death is not yet determined, nor is it known whether global protein glutathionylation increases in other cells types exposed to oxLDL; however, these questions form the basis for future studies.
Nonaka et al.
) discovered that patients with arteriosclerosis of the extremities (i.e.
, arteriosclerosis obliterans, ASO), exhibit increased glutathionylation of serum proteins detected by SDS-PAGE followed by GST overlay. Remarkably, there was a positive correlation between disease progression and magnitude of protein glutathionylation measured, leading the authors to conclude that serum protein glutathionylation is both a sensitive and specific marker of ASO. However, many of the patients enrolled in the study had comorbid conditions also associated with increased serum protein–SSG, such as tobacco use (209
), making the specificity of this marker for ASO unlikely. Importantly, these authors identified the serum protein apoB100 as a target for increased glutathionylation in ASO. ApoB100 is the major component of LDL, and it is tempting to speculate that its glutathionylation could affect its function, as has been shown for other post-translational modifications (292
). Whether apoB100–SSG simply represents a disease marker, or contributes to the pathogenesis of ASO, remains an open question.
In the case of SERCA, the sarcoplasmic calcium ATPase, glutathionylation appears to be part of a normal regulatory mechanism that is disrupted during atherosclerosis. Adachi and colleagues (2
) demonstrated that SERCA glutathionylation occurs in vascular cell lines and tissues in the presence of RNS and endogenous GSH. Glutathionylation could be stimulated by physiological ligands known to generate RNS (e.g.
, acetylcholine, bradykinin), led to increased SERCA ATPase activity, was correlated with vessel dilation, and was resistant to a cGMP inhibitor, leading the authors to propose that SERCA glutathionylation represents a physiological, cGMP-independent mechanism of vessel relaxation. Site-directed mutagenesis and mass spectroscopic analysis suggested that glutathionylation of Cys674, located in the cytosolic-facing hinge domain, was responsible for SERCA activation. Interestingly, analysis of cysteine modifications from atherosclerotic vs.
normal rabbit aortas indicated increased sulfonate formation (including C674), which corresponded to decreased NO-induced relaxation, glutathionylation, and Ca2+
reuptake. Taken together, these observations suggest that irreversible oxidation (i.e.
, sulfonic acid formation) of SERCA's C674 during atherosclerosis prevents regulation of function by reversible glutathionylation and may contribute to the impaired vasodilation response to NO in atherosclerotic smooth muscle.
Adachi et al.
) demonstrated that glutathionylation of Ras may contribute to vascular hypertrophy (implicated in atherosclerosis and hypertension) by activating protein synthesis in rat vascular smooth muscle cells (VSMCs). Treatment of VSMCs with angiotensin II (AII), an established stimulus for vascular hypertrophy, led to glutathionylation and activation of Ras, which resulted in increased phosphorylation of p38 and Akt, and increased protein synthesis (, left). These effects were dependent upon NADPH oxidase activation and ROS formation [shown separately to be activated by AII (162
)], and were blocked by overexpression of Grx1 or mutation of Ras at the site of glutathionylation (C118). Interestingly, AII-stimulated ERK activation, which contributes to AII-induced protein synthesis, was not redox-sensitive and proposed to occur independently of Ras glutathionylation. Like the work of Pimentel et al.
), the work of Adachi and colleagues represents an excellent demonstration of protein regulation by S
-glutathionylation: Ras–SSG formed in response to a physiological stimulus (AII treatment); glutathionylation resulted in a change in protein activity (increased Raf binding); was reversed by Grx1 (via
overexpression); and was correlated to a physiological outcome (protein synthesis).
The work of Adachi and Pimentel point to several common events in hypertrophic signaling within the heart and vasculature: both require production of endogenous H2O2, result in Ras glutathionylation, and activate signaling pathways that ultimately result in increased protein synthesis. However, it is intriguing that the Ras–SSG-activated pathways implicated in hypertrophy differ in the two model systems. Why might H2O2-induced Ras glutathionylation activate the Raf/MEK/ERK pathway in cardiomyocytes vs. p38 and Akt–but not ERK–in vascular smooth muscle? () The answer could reflect differences in signal transduction networking between cell types, different degrees of Ras glutathionylation resulting from each stimulus (assuming different thresholds of activation for downstream pathways), and/or distinct localization of ROS production (and subsequent Ras glutathionylation) depending upon the nature of the stimulus (i.e., NADPH oxidase vs. the source of strain-stimulated ROS).
In addition to modulating AII signaling in VSMCs, Ras-SSG may contribute to atherosclerosis by mediating the response to oxLDL in endothelial cells (). Clavreul et al.
) demonstrated that treatment of bovine aortic endothelial cells (BAECs) with peroxynitrite led to Ras glutathionylation and activation of both ERK and Akt pathways (, left), and some of these observations were recapitulated with oxLDL treatment. Unlike mechanical strain- and AII-induced Ras glutathionylation, which required formation of H2
, oxLDL-mediated glutathionylation was dependent upon peroxynitrite. The authors argue for a thiol–disulfide exchange mechanism with GSSG, based on proposed chemical reactions between peroxynitrite and GSH (producing GSSG), which exhibit a time course of GSSG formation compatible with that observed for Ras glutathionylation. This mechanism is feasible if the Kmix
for Ras-Cys118-SSG formation is similar to the GSH:GSSG ratio achieved during peroxynitrite treatment (98
); but GSSG concentration was not measured in this study, and to the best of our knowledge, the Kmix
for Ras-C118 has not been determined. Therefore, the mechanism of Ras glutathionylation associated with peroxynitrite treatment remains unclear.
FIG. 5. Downstream effects of Ras glutathionylation in response to exogenous peroxinitrite or oxLDL. This figure depicts distinct downstream events resulting from Ras glutathionylation in bovine aortic endothelial cells in response to peroxinitrite added exogenously (more ...)
Specific physiological effects of Ras glutathionylation (and subsequent activation of MEK/ERK and Akt) that might contribute to atherosclerosis were not reported here; however, it has been reported in other contexts that MEK/ERK activation within the endothelium may induce proliferation of endothelial cells, contributing to atherogenic vascular remodeling (238
Although a mechanism for endothelial Ras–SSG-induced atherogenesis was not fully elucidated by Clavreul et al.
), a role for oxLDL-induced Ras glutathionylation in insulin resistance was described in a subsequent study by the same group (48
) (also see section on diabetes). Here, the effects of oxLDL-induced Ras glutathionylation were followed over a longer time course, and cross-talk with a second signaling pathway [insulin/insulin-receptor substrate (IRS)/Akt] was explored. Here, as in the previous study (47
), oxLDL-induced Akt activation was transient, peaking at 15 min; however, ERK activation was sustained (>1
h). Moreover, subsequent activation of Akt by insulin/IRS was blunted by pretreatment with oxLDL, presumably via
ERK-mediated phosphorylation (and inactivation) of IRS (, right). This work enhances understanding of downstream effects of Ras-SSG in endothelial cells, particularly within the context of insulin resistance. However, the distinctive effects of peroxynitrite and oxLDL on the time course of ERK activation raise questions about the use of exogenous
peroxynitrite as a physiologically relevant stimulus.
While studied in different vascular cells using different oxidative stimuli, both Adachi and Clavreul report Ras—mediated phosphorylation of Akt, which is diminished by overexpression of Grx1. Interestingly, Wang et al.
) report an opposite correlation between Grx1 activity and Akt activation in BAECs exposed to laminar flow. Grx1 activity approximately doubled within 5
min of exposure to physiological flow rates, and this activation correlated to increased phosphorylation of Akt and eNOS. Akt and eNOS phosphorylation were augmented with overexpression of Grx1, and diminished after treatment with Grx1 siRNA, suggesting that Grx1 activity precedes their activation, although a specific mechanism was not identified. These observations are consistent with those of Murata et al.
), who reported increased Akt phosphorylation in H9c2 cardiomyoblasts overexpressing Grx1. Taken together, these studies highlight the complex relationship between Grx, protein glutathionylation, and Akt activity within the cardiovascular system. Importantly, Akt is emerging as a complicated signaling molecule within the heart and vasculature, implicated in various pathological signaling events, as well as in normal development and homeostasis (205
). It is conceivable that Grx could participate in regulating the balance between physiological (i.e
., laminar flow-induced) and pathophysiological (i.e
., ATII-induced) Akt activation. Determining the status of Akt activation (as well as downstream effects such as eNOS activity and vessel hypertrophy) in Grx TG and KO animals would help address this complex situation.
An emerging contributor to atherogenesis is tumor necrosis factor-alpha (TNFα), which is thought to induce expression of adhesion molecules on endothelial cells and contribute to vascular smooth muscle cell apoptosis (73
). Pan and Berk (227
) treated endothelial cells with a combination of TNFα and cycloheximide (CHX), and observed Grx activation, pro-caspase-3 deglutathionylation, caspase-3 cleavage, and increased apoptosis. These effects were blocked by transfection with siRNA against Grx1, leading the authors to propose that Grx1-mediated deglutathionylation of pro-caspase-3 contributes to TNFα-induced apoptosis. Importantly, this study was the first to demonstrate glutathionylation of pro-caspase-3 and its effect on susceptibility to cleavage, and this report also identifies caspase-3 as another protein that exists in a glutathionylated state under resting conditions, becoming deglutathionylated by Grx1 in response to an ROS-generating stimulus.
Still, there are some difficulties in interpreting the results of this work. First, are the effects on caspase-3 glutathionylation due to TNFα or CHX? This is difficult to answer because the treatment given to control cells is not explicitly stated. Second, how closely does the TNFα concentration given to BAECs compare to circulating levels in diseased vessels? Here, a dose response curve of response to TNFα would be informative. Finally, this study raises an important question about the potential role of Grx in atheroprotection. In the case of Ras, Grx levels were correlated with decreased Ras–SSG and decreased hypertrophic signaling. In the case of pro-caspase-3, however, Grx overexpression (and deglutathionylation) led to increased apoptosis, a presumably atherogenic event. Taken together, these results highlight the fact that the role of Grx in cardiovascular disease may not be entirely straightforward, with its roles in disease protection or progression dependent upon cell type, extracellular stimuli, etc.