Perturbations of cellular redox couples are instructive in applications that investigate the mechanisms of thiol-based signaling, protection from inflammatory reactive oxygen species and the metabolism of cysteines. Use of small molecule inhibitors such as carmustine (inhibitor of glutathione reductase) or dehydroepiandrosterone (inhibitor of G6PD) provide rapid alterations in the behavior of the redox enzymatic network, but this is potentially at the expense of specificity. Alternatively, small interfering RNA (siRNA) can transiently reduce protein levels with a high degree of specificity, but cellular characterization of the induced changes is often limited by the viability and transduction efficiency of the cells under investigation. Lentiviral infection of shRNA is purported to address both specificity and cell number limitations through the selection for stable incorporation of the interfering RNA within the genome. An additional benefit of stable silencing is the ability to assess long-term changes in the expression levels of other genes in response to the target. These changes take place on time-scales that vary among individual proteins; for example, in a genome-wide study of mouse embryonic stem cells, mRNA of NAD(P)H oxidase isoforms decay with half-lives on the order of 4-5 hours, while peroxiredoxins 1 & 2 and isocitrate dehydrogenase 1 & 2 had half-lives of 20-24 hours . Consequently, when accounting for changes in mRNA and protein decay rates and sequential feedback, cellular remodeling in response to a silenced redox protein may not be fully implemented across all proteins to a new homeostatic state for several days. All of the shRNA cell lines used for this study had undergone at least three passages from the lentiviral infection and puromycin selection before characterization.
Because of the advantages to using shRNA, this method of examining redox regulation of cellular processes has been increasingly used to study effects of NAD(P)H supply, glutaredoxin, thioredoxin, and associated reductases [16–22]. Many of these studies rely on perturbation of the target protein in order to observe a phenotypic change, such as sensitivity to diamide-induced oxidative stress  or increased cellular senescence , and rely on the assumption that the rest of the antioxidant enzyme system remains intact. Our results, however, question this assumption by the changes observed in both the empty lentiviral construct and off-target changes due to specific protein silencing. We measured dramatic changes in redox protein mRNA levels due to the presence of the empty lentiviral vector, compared to uninfected cells. This is consistent with a vector-independent interferon response that is initiated by shRNA  or altered regulation of autocrine cytokines . Furthermore, interferon γ stimulated response elements (ISRE) have been identified in the promoter regions of Nox1 and Nox2 [28, 29] and therefore may potentially also control expression levels of other proteins involved in regulation of ROS, such as Duox1 and Rac1. Grx1 has been reported to be an essential regulator of interferon response factor-3 in Sendai virus infected cells ; if this role is conserved in all lentiviral infection, this mechanism may partially account for the sensitivity of the Jurkat cells to Grx1-specific RNA interference. Phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2) through protein kinase R (PKR) is induced by IFN-γ and TNF-α [31, 32] as well as a variety of stress conditions, including viral infection, and has been shown to down-regulate protein synthesis [33, 34]. Therefore, lentiviral infection could potentially have post-translational effects on eIF2-α, thereby altering the regulation of protein synthesis. It should also be noted that treatment with aminoglycoside antibiotics has been shown to have effects on the expression levels of Grx1, and oxidative stress induced by antibiotics should be included as a potential cause for the changes we observed . We have taken this into account in our experiments by using a control cell line (pLKO vector cells) that was cultured in the same puromycin-containing media as our shRNA cells. We compared our shRNA cells to this cell line and also compared mRNA levels in our shRNA and pLKO cell lines to that of wild-type cells (Figure 2, Additional file 1); however, a direct comparison of the effects of puromycin was not feasible, as puromycin was toxic to our wild-type cells.
In past cDNA microarray studies of responses associated with oxidative stress, transcriptional changes are reported as up- or down-regulated expression levels rather than the coordinated regulation of the redox-related enzymes [36–39]. Prior microarray studies on virally-infected T cells have not reported significant changes across the antioxidant enzyme network as observed in the present analysis, instead yielding one or two redox-related genes in the genomic arrays [40, 41]. In contrast, other studies have demonstrated careful characterization of antioxidant enzyme changes, but only on a select few proteins (e.g. ). Systematic histological characterization of redox protein distribution provides complementary information, but does not allow for direct multivariate expression relationships on a large scale . The intent of this study was to provide a more targeted approach with proteins that coordinate H2O2 production and clearance from the intracellular environment while still monitoring enough genes to observe systemic effects. Microarray and qRT PCR results may not always directly correspond to protein levels; however for a subset of proteins characterized in the present data (Grx1, Trx1, G6PD, Prx2, and Duox1) a linear relationship between mRNA and protein levels was observed.
Peroxiredoxins have emerged as important regulators of cellular redox status [1, 44]. Among the coupled reactions of peroxiredoxin, thioredoxin, and sulfiredoxin, our network approach yielded novel insight into the regulation of this enzyme family (Figure 4A). A strong co-expression pattern emerged between the three peroxiredoxins measured (Prx1, 2, 4). Thioredoxin reductase also tightly co-varied with the peroxiredoxins while Trx1 showed less relation. A direct correlation between Srx1 and the rest of the peroxiredoxin branch was not established across the cell lines assayed, but the Trx1 knockdown cells showed a significant decrease in Srx1 (35%, p < 0.05). The off-target Srx1 compensation caused by attenuation of thioredoxin 1 expression may be more tightly regulated than our analysis suggests and could be explored further by epi-allelic strains of Trx1 shRNA targets. The distance between Prx2 and Grx1 in the principal component analysis demonstrated a stronger expression regulation between these two proteins than with Srx1 or Trx1. Because glutaredoxin 1 is not a substrate for the peroxiredoxins , this trend was unexpected but it was supported by analysis with additional cell lines. It has been postulated by Winterbourn and Hampton that additional functionality of peroxiredoxins through facilitated, indirect oxidation of other proteins could provide an alternate method of oxidative signal transmission ; if protein sulfenic acid is generated in this manner and subsequently protected by S-glutathionylation, than coordination between peroxiredoxins and Grx1 expression would be appropriate.
Disequilibrium of redox couples between the cytoplasm, ER, mitochondria, and nuclei has been elucidated through investigations of the differential subcellular sensitivities to oxidative stress [2, 47–54]. The nature of the oxidative stress preferentially impacts different locations ; for example, EGF signaling induces Trx1 oxidation , while Trx2 is preferentially oxidized with TNF-α treatment . The co-varying relationships and expression patterns illustrated in Figure 4A indicate that this insularity extends to a lack of up-regulated compensation of alternate isoforms when a cytosolic component (Trx1, Grx1) is altered, as we observed no statistical differences in the corresponding mitochondrial Trx2 and Grx2. In contrast, the computational modeling results suggest changes in the cytosolic NAD(P)H supply by G6PD silencing may result in enhanced mitochondrial NAD(P)H production by a dehydrogenase, IDH2. A significant decrease (35%, p < 0.05) in Trx2 upon Grx1 silencing was detected, however, but no known mechanism for communication between these compartmentalized redox proteins is known.
Changes due to the presence of the lentivirus could be accounted for with proper control experiments if not for the added non-specific off-target effects resulting from manipulating the various redox couples through RNA interference. The shRNA sequences used in the present study were for disparate protein targets with unique sequences, thus the changes observed are attributed to compensation by the cell for decreased reducing capabilities rather than direct silencing by RNA interference. Computational analysis allowed for the direct comparison of the idealized shRNA specificity to the more likely behavior if changes in mRNA levels reflect changes in protein translation. Although all the cell lines with lentivirus except the Grx1 shRNA knockdown cell line showed insignificant differences in antioxidant capacity due to off-target effects, it should be noted that not all the off-target effects detected by qRT PCR were capable of being simulated using the current cytosolic model. For example, downregulation of oxidative enzymes such as the Nox/Duox family, which would likely impact signaling-induced antioxidative responses, is not reflected in the models. This feature of ROS production is missing as the computational description only accounts for the dissipation of H2O2 by permeation through the cell membrane and clearance by glutathione, peroxisomes, and cytosolic enzymatic reactions. Our data shows that Duox1 was significantly downregulated in both the Grx1 and G6PD shRNA cell lines. It is possible that a more comprehensive model, one which is spatially descriptive and includes mitochondrial and membrane related antioxidant enzymes, could more readily show the resulting effect of off-target alterations on cellular antioxidant capacity. However, with the current modeling analysis, it is clear that off-target effects involving the G6PD enzyme system are more likely to result in significant changes in the overall redox capacity of the cell, compared to the idealized knockdown condition.
A caveat of our simulations is the numerous metabolic and post-translational modifications that are likely occurring in the cell lines. Although we maintained uniform redox state of NADPH/NADP+, Trx1-SH2/Trx1-SS, and GSH/GSSG for each cell-line specific model, each of these may be altered in response to the lentiviral constructs. For instance, G6PD knockdowns cells have been shown to exhibit an impaired ability to regenerate GSH . Another caveat is that we have assumed that changes in mRNA levels will be reflected in protein levels. Although our detailed analysis of two proteins did show a linear correlation between mRNA and protein (Figure 3), this relation may not be upheld for all proteins due to translational regulation. Furthermore, proteins beyond the scope of our array involved in transport and metabolite synthesis may be altered to control the total available pools of NADPH and glutathione. Our analysis, therefore, is useful strictly as a means of examining one aspect of redox remodeling associated with viral infection and shRNA.
A surprising finding of our experimental and computational work is the dichotomy between the numerous changes that result at the mRNA level due to the introduction of lentiviral mRNA and the minor possible functional consequences in the overall ability of cells to handle acute, exogenous oxidative insult. Our shRNA perturbations of each redox couple indicated sensitivity to protein levels that were apparent only by inspection of multiple metrics of oxidative stress (Figure 5). With the exception of the Grx1/G6PD relationship, the off-target effects largely did not influence functionality of the redox enzymatic network as tested by our simulations. These results need to be investigated experimentally; for example, future studies of combined targeted silencing of both genes could characterize the necessary ratios of each protein required to maintain a robust redox system. Additional experiments are also necessary to establish whether the concomitant downregulation of ROS producing enzymes and enzymes involved in ROS scavenging is reflected in an increased resistance to cellular oxidation required for many signal transduction processes.