Evaluation of the salivary glutaredoxin-1 levels in oral lichen planus and oral squamous cell carcinoma.

BACKGROUND: Glutaredoxin-1, as a component of antioxidant system, plays a crucial role in pathogenesis of some cancers and pre-malignant lesions. The aim of this study is to assess salivary levels of glutaredoxin-1 in oral lichen planus (OLP) and oral squamous cell carcinoma (OSCC) patients compared with healthy controls. METHODS & MATERIALS: This cross-sectional study was conducted on 28 OLP patients, 20 OSCC cases, and 40 healthy subjects. Their salivary glutaredoxin-1 was assessed by the enzyme-linked immunosorbent assay (ELISA). RESULTS: Regarding the glutaredoxin-1 level, there was no significant difference between the three studied groups (p=0.073); however, the salivary glutaredoxin-1levels were higher in the healthy subjects than the OLP and OSCC patients. Among OSCC patients, the salivary glutaredoxin-1 levels were significantly higher in the males (p=0.03). According to glutaredoxin-1 levels, the difference between keratotic and non-keratotic OLP lesions were not significant (p=0.98). Furthermore, there was no significant difference between various clinical manifestations and grades of OSCC, (p=0.08, p=0.56, respectively). CONCLUSION: Salivary glutaredoxin-1 levels were lower in OLP and OSCC patients compared to the normal volunteers; therefore the role of glutaredoxin-1 in the pathogenesis of these diseases could be proposed.

Genistein-Calcitriol Mitigates Hyperosmotic Stress-Induced TonEBP, CFTR Dysfunction, VDR Degradation and Inflammation in Dry Eye Disease.

Dry eye disease (DED) signs and symptoms are causally associated with increased ocular surface (OS) inflammation. Modulation of key regulators of aberrant OS inflammation is of interest for clinical management. We investigated the status and the potential to harness key endogenous protective factors, such as cystic fibrosis transmembrane conductance regulator (CFTR) and vitamin D receptor (VDR) in hyperosmotic stress-associated inflammation in patients with DED and in vitro. Conjunctival impression cytology samples from control subjects (n = 11) and patients with DED (n = 15) were used to determine the status of hyperosmotic stress (TonEBP/NFAT5), inflammation (IL-6, IL-8, IL-17A/F, TNFα, MMP9, and MCP1), VDR, and intracellular chloride ion (GLRX5) by quantitative polymerase chain reaction and/or immunofluorescence. Human corneal epithelial cells (HCECs) were used to study the effect of CFTR activator (genistein) and vitamin D (calcitriol) in hyperosmotic stress (HOs)-induced response in vitro. Western blotting was used to determine the expression of these proteins, along with p-p38. Significantly, higher expression of inflammatory factors, TonEBP, GLRX5, and reduced VDR were observed in patients with DED and in HOs-induced HCECs in vitro. Expression of TonEBP positively correlated with expression of inflammatory genes in DED. Increased TonEBP and GLRX5 provides confirmation of osmotic stress and chloride ion imbalance in OS epithelium in DED. These along with reduced VDR suggests dysregulated OS homeostasis in DED. Combination of genistein and calcitriol reduced HOs-induced TonEBP, inflammatory gene expression, and p-p38, and abated VDR degradation in HCECs. Henceforth, this combination should be further explored for its relevance in the management of DED.

Dual-color 3D-dSTORM colocalization and quantification of ROXY1 and RNAPII variants throughout the transcription cycle in root meristem nuclei.

To unravel the function of a protein of interest, it is crucial to asses to what extent it associates via direct interactions or by overlapping expression with other proteins. ROXY1, a land plant-specific glutaredoxin, exerts a function in Arabidopsis flower development and interacts with TGA transcription factors in the nucleus. We detected a novel ROXY1 function in the root meristem. Root cells that lack chlorophyll reducing plant-specific background problems that can hamper colocalization 3D microscopy. Thus far, a super-resolution three-dimensional stochastic optical reconstruction microscopy (3D-dSTORM) approach has mainly been applied in animal studies. We established 3D-dSTORM using the roxy1 mutant complemented with green fluorescence protein-ROXY1 and investigated its colocalization with three distinct RNAPII isoforms. To quantify the colocalization results, 3D-dSTORM was coupled with the coordinate-based colocalization method. Interestingly, ROXY1 proteins colocalize with different RNA polymerase II (RNAPII) isoforms that are active at distinct transcription cycle steps. Our colocalization data provide new insights on nuclear glutaredoxin activities suggesting that ROXY1 is not only required in early transcription initiation events via interaction with transcription factors but likely also participates throughout further transcription processes until late termination steps. Furthermore, we showed the applicability of the combined approaches to detect and quantify responses to altered growth conditions, exemplified by analysis of H2 O2 treatment, causing a dissociation of ROXY1 and RNAPII isoforms. We envisage that the powerful dual-color 3D-dSTORM/coordinate-based colocalization combination offers plant cell biologists the opportunity to colocalize and quantify root meristem proteins at an increased, unprecedented resolution level <50 nm, which will enable the detection of novel subcellular protein associations and functions.

Glutaredoxin1 Diminishes Amyloid Beta-Mediated Oxidation of F-Actin and Reverses Cognitive Deficits in an Alzheimer's Disease Mouse Model.

Aims: Reactive oxygen species (ROS) generated during Alzheimer's disease (AD) pathogenesis through multiple sources are implicated in synaptic pathology observed in the disease. We have previously shown F-actin disassembly in dendritic spines in early AD (34). The actin cytoskeleton can be oxidatively modified resulting in altered F-actin dynamics. Therefore, we investigated whether disruption of redox signaling could contribute to actin network disassembly and downstream effects in the amyloid precursor protein/presenilin-1 double transgenic (APP/PS1) mouse model of AD. Results: Synaptosomal preparations from 1-month-old APP/PS1 mice showed an increase in ROS levels, coupled with a decrease in the reduced form of F-actin and increase in glutathionylated synaptosomal actin. Furthermore, synaptic glutaredoxin 1 (Grx1) and thioredoxin levels were found to be lowered. Overexpressing Grx1 in the brains of these mice not only reversed F-actin loss seen in APP/PS1 mice but also restored memory recall after contextual fear conditioning. F-actin levels and F-actin nanoarchitecture in spines were also stabilized by Grx1 overexpression in APP/PS1 primary cortical neurons, indicating that glutathionylation of F-actin is a critical event in early pathogenesis of AD, which leads to spine loss. Innovation: Loss of thiol/disulfide oxidoreductases in the synapse along with increase in ROS can render F-actin nanoarchitecture susceptible to oxidative modifications in AD. Conclusions: Our findings provide novel evidence that altered redox signaling in the form of S-glutathionylation and reduced Grx1 levels can lead to synaptic dysfunction during AD pathogenesis by directly disrupting the F-actin nanoarchitecture in spines. Increasing Grx1 levels is a potential target for novel disease-modifying therapies for AD.

Dimers of glutaredoxin 2 as mitochondrial redox sensors in selenite-induced oxidative stress.

Glutaredoxin 2 (Grx2) has been previously shown to link thioredoxin and glutathione systems receiving reducing equivalents by both thioredoxin reductase and glutathione. Grx2 catalyzes protein glutathionylation/de-glutathionylation and can coordinate an iron-sulfur cluster, forming inactive dimers stabilized by two molecules of glutathione. This protein is mainly located in the mitochondrial matrix, though other isoforms have been found in the cytosolic and nuclear cell compartments. In the present study, we have analyzed the monomeric and dimeric states of Grx2 under different redox conditions in HeLa cells, and sodium selenite was utilized as the principal oxidizing agent. After selenite treatment, an increased glutathione oxidation was associated to Grx2 monomerization and activation, specifically in the mitochondrial compartment. Interestingly, in mitochondria, a large decline of thioredoxin reductase activity was also observed concomitantly to Grx2 activity stimulation. In addition, Grx2 monomerization led to an increase free iron ions concentration in the mitochondrial matrix, induction of lipid peroxidation and decrease of the mitochondrial membrane potential, indicating that the disassembly of Grx2 dimer involved the release of the iron-sulfur cluster in the mitochondrial matrix. Moreover, sodium selenite-triggered lipid and protein oxidation was partially prevented by deferiprone, an iron chelator with mitochondriotropic properties, suggesting a role of the iron-sulfur cluster release in the observed impairment of mitochondrial functions. Thus, by sensing the overall cellular redox conditions, mitochondrial Grx2 dimers become active monomers upon oxidative stress induced by sodium selenite with the consequent release of the iron-sulfur cluster, leading to activation of the intrinsic apoptotic pathway.

Stable Integration and Comparison of hGrx1-roGFP2 and sfroGFP2 Redox Probes in the Malaria Parasite Plasmodium falciparum.

Studying redox metabolism in malaria parasites is of great interest for understanding parasite biology, parasite-host interactions, and mechanisms of drug action. Genetically encoded fluorescent redox sensors have recently been described as powerful tools for determining the glutathione-dependent redox potential in living parasites. In the present study, we genomically integrated and expressed the ratiometric redox sensors hGrx1-roGFP2 (human glutaredoxin 1 fused to reduction-oxidation sensitive green fluorescent protein) and sfroGFP2 (superfolder roGFP2) in the cytosol of NF54- attB blood-stage Plasmodium falciparum parasites. Both sensors were evaluated in vitro and in cell culture with regard to their fluorescence properties and reactivity. As genomic integration allows for the stable expression of redox sensors in parasites, we systematically compared single live-cell imaging with plate reader detection. For these comparisons, short-term effects of redox-active compounds were analyzed along with mid- and long-term effects of selected antimalarial agents. Of note, the single components of the redox probes themselves did not influence the redox balance of the parasites. Our analyses revealed comparable results for both the hGrx1-roGFP2 and sfroGFP2 probes, with sfroGFP2 exhibiting a more pronounced fluorescence intensity in cellulo. Accordingly, the sfroGFP2 probe was employed to monitor the fluorescence signals throughout the parasites' asexual life cycle. Through the use of stable genomic integration, we demonstrate a means of overcoming the limitations of transient transfection, allowing more detailed in-cell studies as well as high-throughput analyses using plate reader-based approaches.

Exosomes from patients with septic shock convey miRNAs related to inflammation and cell cycle regulation: new signaling pathways in sepsis?

BACKGROUND: Exosomes isolated from plasma of patients with sepsis may induce vascular apoptosis and myocardial dysfunction by mechanisms related to inflammation and oxidative stress. Despite previous studies demonstrating that these vesicles contain genetic material related to cellular communication, their molecular cargo during sepsis is relatively unknown. In this study, we evaluated the presence of microRNAs (miRNAs) and messenger RNAs (mRNAs) related to inflammatory response and redox metabolism in exosomes of patients with septic shock. METHODS: Blood samples were collected from 24 patients with septic shock at ICU admission and after 7 days of treatment. Twelve healthy volunteers were used as control subjects. Exosomes were isolated by ultracentrifugation, and their miRNA and mRNA content was evaluated by qRT-PCR array. RESULTS: As compared with healthy volunteers, exosomes from patients with sepsis had significant changes in 65 exosomal miRNAs. Twenty-eight miRNAs were differentially expressed, both at enrollment and after 7 days, with similar kinetics (18 miRNAs upregulated and 10 downregulated). At enrollment, 35 differentially expressed miRNAs clustered patients with sepsis according to survival. The pathways enriched by the miRNAs of patients with sepsis compared with control subjects were related mostly to inflammatory response. The comparison of miRNAs from patients with sepsis according to hospital survival demonstrated pathways related mostly to cell cycle regulation. At enrollment, sepsis was associated with significant increases in the expression of mRNAs related to redox metabolism (myeloperoxidase, 64-fold; PRDX3, 2.6-fold; SOD2, 2.2-fold) and redox-responsive genes (FOXM1, 21-fold; SELS, 16-fold; GLRX2, 3.4-fold). The expression of myeloperoxidase mRNA remained elevated after 7 days (65-fold). CONCLUSIONS: Exosomes from patients with septic shock convey miRNAs and mRNAs related to pathogenic pathways, including inflammatory response, oxidative stress, and cell cycle regulation. Exosomes may represent a novel mechanism for intercellular communication during sepsis.

Distinct responses of compartmentalized glutathione redox potentials to pharmacologic quinones targeting NQO1.

Deoxynyboquinone (DNQ), a potent novel quinone-based antineoplastic agent, selectively kills solid cancers with overexpressed cytosolic NAD(P)H:quinone oxidoreductase-1 (NQO1) via excessive ROS production. A genetically encoded redox-sensitive probe was used to monitor intraorganellar glutathione redox potentials (EGSH) as a direct indicator of cellular oxidative stress following chemotherapeutic administration. Beta-lapachone (ß-lap) and DNQ-induced spatiotemporal redox responses were monitored in human lung A549 and pancreatic MIA-PaCa-2 adenocarcinoma cells incubated with or without dicumarol and ES936, potent NQO1 inhibitors. Immediate oxidation of EGSH in both the cytosol and mitochondrial matrix was observed in response to DNQ and ß-lap. The DNQ-induced cytosolic oxidation was fully prevented with NQO1 inhibition, whereas mitochondrial oxidation in A549 was NQO1-independent in contrast to MIA-PaCa-2 cells. However, at pharmacologic concentrations of ß-lap both quinone-based substrates directly oxidized the redox probe, a possible sign of off-target reactivity with cellular thiols. Together, these data provide new evidence that DNQ's direct and discerning NQO1 substrate specificity underlies its pharmacologic potency, while ß-lap elicits off-target responses at its effective doses.

The chimeric transcript RUNX1-GLRX5: a biomarker for good postoperative prognosis in Stage IA non-small-cell lung cancer.

Stage IA non-small-cell lung cancer cases have been recognized as having a low risk of relapse; however, occasionally, relapse may occur. To predict clinical outcome in Stage IA non-small-cell lung cancer patients, we searched for chimeric transcripts that can be used as biomarkers and identified a novel chimeric transcript, RUNX1-GLRX5, comprising RUNX1, a transcription factor, and GLRX5. This chimera was detected in approximately half of the investigated Stage IA non-small-cell lung cancer patients (44/104 cases, 42.3%). Although there was no significant difference in the overall survival rate between RUNX1-GLRX5-positive and -negative cases (P = 0.088), a significantly lower relapse rate was observed in the RUNX1-GLRX5-positive cases (P = 0.039), indicating that this chimera can be used as a biomarker for good prognosis in Stage IA patients. Detection of the RUNX1-GLRX5 chimeric transcript may therefore be useful for the determination of a postoperative treatment plan for Stage IA non-small-cell lung cancer patients.

Expression of thioredoxins and glutaredoxins in human hepatocellular carcinoma: correlation to cell proliferation, tumor size and metabolic syndrome.

Thioredoxins (Trx) and glutaredoxins (Grx) are thiol oxidoreductases that are ubiquitously expressed, and are involved in several biological processes. The expression of thioredoxins and glutaredoxins is induced in many neoplasms, and correlates with prognosis in gallbladder and colorectal carcinoma. The aim of the present study was to examine the expression pattern of these proteins (redoxins) in hepatocellular carcinoma (HCC) and to correlate their levels with clinical features. Paraffin-embedded tissues from 25 patients resected for HCC and 15 patients resected for colorectal carcinoma (CRC) liver metastases were analyzed with immunohistochemistry. Our results showed that Trx1, Trx2 and Grx5 were upregulated in HCCs as compared to the respective surrounding liver. In comparison, almost all redoxins were upregulated in CRC liver metastases, with Trx1 and Grx3 being significantly more increased in the CRC liver metastases than in the primary HCC tumors. In HCC, Trx1 correlated significantly with cell proliferation, and with a trend towards increased levels with micro-vascular invasion, while expression of Trx2 decreased with tumor size. Trx1 levels were lower in tumors of males, smokers, and patients with high alcohol consumption. Grx2 levels were significantly higher in patients with metabolic syndrome. In conclusion, this study illustrates specific correlations of individual redoxins to clinical features of HCC, and implicates the redoxins in the pathogenesis of HCC.

Inhibition of glutathione synthesis distinctly alters mitochondrial and cytosolic redox poise.

The glutathione couple GSH/GSSG is the most abundant cellular redox buffer and is not at equilibrium among intracellular compartments. Perturbation of glutathione poise has been associated with tumorigenesis; however, due to analytical limitations, the underlying mechanisms behind this relationship are poorly understood. In this regard, we have implemented a ratiometric, genetically encoded redox-sensitive green fluorescent protein fused to human glutaredoxin (Grx1-roGFP2) to monitor real-time glutathione redox potentials in the cytosol and mitochondrial matrix of tumorigenic and non-tumorigenic cells. First, we demonstrated that recovery time in both compartments depended upon the length of exposure to oxidative challenge with diamide, a thiol-oxidizing agent. We then monitored changes in glutathione poise in cytosolic and mitochondrial matrices following inhibition of glutathione (GSH) synthesis with L-buthionine sulphoximine (BSO). The mitochondrial matrix showed higher oxidation in the BSO-treated cells indicating distinct compartmental alterations in redox poise. Finally, the contributory role of the p53 protein in supporting cytosolic redox poise was demonstrated. Inactivation of the p53 pathway by expression of a dominant-negative p53 protein sensitized the cytosol to oxidation in BSO-treated tumor cells. As a result, both compartments of PF161-T+p53(DD) cells were equally oxidized ≈20 mV by inhibition of GSH synthesis. Conversely, mitochondrial oxidation was independent of p53 status in GSH-deficient tumor cells. Taken together, these findings indicate different redox requirements for the glutathione thiol/disulfide redox couple within the cytosol and mitochondria of resting cells and reveal distinct regulation of their redox poise in response to inhibition of glutathione biosynthesis.

Molecular characterization of glutaredoxin 2 from Ostrinia furnacalis.

Glutaredoxins (GRXs) play very important roles in maintaining intracellular redox homeostasis. In the present study, the full-length cDNA sequence encoding GRX2, named OfurGRX2 (GenBank accession no. GU393246), was obtained from Ostrinia furnacalis, using reverse transcription polymerase chain reaction and rapid amplification of cDNA ends. Sequence analysis revealed that the open reading frame of OfurGRX2 consists of 351 nucleotides encoding 116 amino acid residues with a predicted molecular weight of 12.6 kDa. Homolog research revealed that OfurGRX2 shares a common active site, CPYC/CPFC, with other insect counterparts. Expression profiles revealed that OfurGRX2 is a ubiquitous gene expressed in insect heads, fat bodies, epidermises, mid guts and muscles. The OfurGRX2 transcript peaked in 36-h larvae of 4th instars, and then suddenly declined in the molting stage. Hormone treatment experiments revealed that 20-hydroxyecodyson (20e) significantly induces the expression of the OfurGRX2 transcript, whereas juvenile hormone (JH) counteracts 20e effects. Adverse stress factors (including starvation, ultraviolet light, mechanical injury, Escherichia coli exposure, and high and low temperatures) dramatically induced OfurGRXGRX2 transcript expression, which confirmed for the first time that GRX2 play important roles in insecta during exposure to adverse environments.

Expression of glutaredoxin-1 in nasal polyps and airway epithelial cells.

BACKGROUND: Glutaredoxins (GRX)-1 is glutathione-dependent oxidoreductase. However, the role of these enzymes remains unknown in airway inflammatory diseases. Therefore, we aimed to establish the expression pattern of GRX-1 in the nasal polyps (NPs) and to assess the regulatory mechanisms associated with GRX-1 expression in interleukin (IL)-1 beta-treated airway epithelial cells. METHODS: The expression of GRX-1 in NPs and normal nasal mucosa were analyzed by reverse-transcription polymerase chain reaction and immunohistochemical staining. IL-1 beta-induced reactive oxygen species (ROS) formation and GRX-1 expression in the airway epithelial cells was determined by flow cytometry and immunoassay. RESULTS: The expression level of GRX-1 in NPs was significantly higher than in the normal nasal mucosa (p < 0.05). GRX-1 was highly expressed in the surface epithelial cells and the submucosal glandular cells in the NPs. IL-1 beta increased the intracellular ROS formation and GRX-1 expression in airway epithelial cells. The inhibition of IL-1 beta-induced ROS production by N-acetyl-cystein, an ROS scavenger, reduced GRX-1 expression. Diphenyleneiodonium and apocynin, NADPH oxidase inhibitors, did not abolish IL-1 beta-induced ROS formation and GRX-1 expression, whereas budesonide attenuated it. CONCLUSION: High GRX-1 expression in NPs might be a primary defense against chronic inflammatory oxidative stress in nasal mucosa. IL-1 beta-induced up-regulation of GRX-1 in airway epithelial cells is probably mediated by ROS. Glucocorticoids can regulate IL-1 beta-induced ROS formation and GRX-1 expression.

Nuclear activity of ROXY1, a glutaredoxin interacting with TGA factors, is required for petal development in Arabidopsis thaliana.

Glutaredoxins (GRXs) have thus far been associated mainly with redox-regulated processes participating in stress responses. However, ROXY1, encoding a GRX, has recently been shown to regulate petal primorida initiation and further petal morphogenesis in Arabidopsis thaliana. ROXY1 belongs to a land plant-specific class of GRXs that has a CC-type active site motif, which deviates from ubiquitously occurring CPYC and CGFS GRXs. Expression studies of yellow fluorescent protein-ROXY1 fusion genes driven by the cauliflower mosaic virus 35S promoter reveal a nucleocytoplasmic distribution of ROXY1. We demonstrate that nuclear localization of ROXY1 is indispensable and thus crucial for its activity in flower development. Yeast two-hybrid screens identified TGA transcription factors as interacting proteins, which was confirmed by bimolecular fluorescence complementation experiments showing their nuclear interaction in planta. Overlapping expression patterns of ROXY1 and TGA genes during flower development demonstrate that ROXY1/TGA protein interactions can occur in vivo and support their biological relevance in petal development. Deletion analysis of ROXY1 demonstrates the importance of the C terminus for its functionality and for mediating ROXY1/TGA protein interactions. Phenotypic analysis of the roxy1-2 pan double mutant and an engineered chimeric repressor mutant from PERIANTHIA (PAN), a floral TGA gene, supports a dual role of ROXY1 in petal development. Together, our results show that the ROXY1 protein functions in the nucleus, likely by modifying PAN posttranslationally and thereby regulating its activity in petal primordia initiation. Additionally, ROXY1 affects later petal morphogenesis, probably by modulating other TGA factors that might act redundantly during differentiation of second whorl organs.

Identification, expression pattern, and characterization of mouse glutaredoxin 2 isoforms.

Glutaredoxin 2 (Grx2) is a glutathione-dependent oxidoreductase involved in the maintenance of mitochondrial redox homeostasis. Grx2 was first characterized as mitochondrial protein, but alternative mRNA variants lacking the transit peptide-encoding first exon were demonstrated for human and proposed for mouse. We systematically screened for alternative transcript variants of mouse Grx2. We identified a total of six exons, three constitutive (II, III, and IV), two alternative first exons (exons Ia and Ic), and one single-cassette exon (exon IIIb) located between exons III and IV. Exons Ic and IIIb are not present in the human genome; mice lack human exon Ib. The six exons give rise to five transcript variants that encode three protein isoforms: mitochondrial Grx2a, a cytosolic isoform that is homologous to the cytosolic/nuclear human Grx2c and present in specific cells of many tissues and the testis-specific isoform Grx2d that is unique to mice. Mouse Grx2c can form an iron/sulfur cluster-bridged dimer, is enzymatically active as a monomer, and can donate electrons to ribonucleotide reductase. Testicular cells lack mitochondrial Grx2a but contain cytosolic Grx2. Prominent immunostaining was detected in spermatogonia and spermatids. These results provide evidence for additional functions of Grx2 in the cytosol, in cell proliferation, and in cellular differentiation.

AtGRX4, an Arabidopsis chloroplastic monothiol glutaredoxin, is able to suppress yeast grx5 mutant phenotypes and respond to oxidative stress.

Arabidopsis monothiol glutaredoxin (Grx), AtGRX4, was targeted to chloroplasts/plastids and had high similarity to yeast Grx5. In yeast expression assays, AtGRX4 localized to the mitochondria and suppressed the sensitivity of grx5 cells to oxidants. In addition, AtGRX4 reduced iron accumulation and rescued the lysine auxotrophy of grx5 cells. In planta, AtGRX4 RNA transcripts accumulated in growing tissues. Furthermore, AtGRX4expression was altered under various stresses. Genetic analysis revealed that seedlings of atgrx4 mutants were sensitive to oxidants. Taken together, these results suggest that AtGRX4 may have important functions in plant growth and development under extreme environments.