Implication of Thioredoxin 1 and Glutaredoxin 1 in H2O2-induced Phosphorylation of JNK and p38 MAP Kinases.

BACKGROUND: Aerobic organisms continuously generate small amounts of Reactive Oxygen Species (ROS), which are involved in the oxidation of sensitive cysteine residues in proteins, leading to the formation of disulfide bonds. Thioredoxin (Trx1) and Glutaredoxin (Grx1) represent key antioxidant enzymes reducing disulfide bonds. OBJECTIVE: In this work, we have focused on the possible protective effect of Trx1 and Grx1 against oxidative stress-induced DNA damage and apoptosis-signaling, by studying the phosphorylation of MAP kinases. METHODS: Trx1 and Grx1 were overexpressed or silenced in cultured H1299 non-small cell lung cancer epithelial cells. We examined cell growth, DNA damage, and the phosphorylation status of MAP kinases following treatment with H2O2. RESULTS: Overexpression of both Trx1 and Grx1 had a significant impact on the growth of H1299 cells and provided protection against H2O2-induced toxicity, as well as acute DNA single-strand breaks. Conversely, silencing of these proteins exacerbated DNA damage. Furthermore, overexpression of Trx1 and Grx1 inhibited the rapid phosphorylation of JNK (especially at 360 min of treatment, ****p=0.004 and **p=0.0033 respectively) and p38 MAP kinases (especially at 360 min of treatment, ****p<0.0001 and ***p=0.0008 respectively) during H2O2 exposure, while their silencing had the opposite effect (especially at 360 min of treatment, ****p<0.0001). CONCLUSION: These results suggest that both Trx1 and Grx1 have protective roles against H2O2 induced toxicity, emphasizing their significance in mitigating oxidative stress-related cellular damage.

Deregulation of the Kallikrein Protease Family in the Salivary Glands of the Sjögren's Syndrome ERdj5 Knockout Mouse Model.

Introduction: The purpose of this study was to identify differentially expressed proteins in salivary glands of the ERdj5 knockout mouse model for Sjögren's syndrome and to elucidate possible mechanisms for the morbid phenotype development. At the same time, we describe for the first time the sexual dimorphism of the murine submandibular salivary gland at the proteome level. Methods: We performed Liquid Chromatography/Mass Spectrometry in salivary gland tissues from both sexes of ERdj5 knockout and 129SV wildtype mice. The resulting list of proteins was evaluated with bioinformatic analysis and selected proteins were validated by western blot and immunohistochemistry and further analyzed at the transcription level by qRT-PCR. Results: We identified 88 deregulated proteins in females, and 55 in males in wildtype vs knockout comparisons. In both sexes, Kallikrein 1b22 was highly upregulated (fold change>25, ANOVA p<0.0001), while all other proteases of this family were either downregulated or not significantly affected by the genotype. Bioinformatic analysis revealed a possible connection with the downregulated NGF that was further validated by independent methods. Concurrently, we identified 416 proteins that were significantly different in the salivary gland proteome of wildtype female vs male mice and highlighted pathways that could be driving the strong female bias of the pathology. Conclusion: Our research provides a list of novel targets and supports the involvement of an NGF-mediating proteolytic deregulation pathway as a focus point towards the better understanding of the underlying mechanism of Sjögren's syndrome.

Sizing Up Extracellular DNA: Instant Chromatin Discharge From Cells When Placed in Serum-Free Conditions.

How do you wash cells? Three out of four of our colleagues use experimental procedures during everyday lab-bench work that can severely impair data interpretation depending on how cells are handled. We show here that a subpopulation (2-3%) of human leukocytes immediately induce a yet unclassified lytic cell death, concomitant with discharge of chromatin entities and cell elimination, when placed in protein-free solutions (i.e., PBS and HBSS). DNA release was not restricted to hematopoietic cells but occurred also in HEK293T cells. Albumin, fetal bovine serum, polyethylene glycol, and Pluronic F-68 supplements prevented chromatin discharge. Expelled chromatin was devoid of surrounding membranes but maintained its original nuclear shape, although ∼10 times enlarged. These structures differed from DNA appearance after osmotic or detergent-induced cell lysis. Besides sounding a cautionary note to the neutrophil extracellular trap (NET) research community, in which ∼50% of all published studies used protein-free media for NET-formation, our study also provides a rapid tool for analysis of chromatin organization.

AAV-mediated ERdj5 overexpression protects against P23H rhodopsin toxicity.

Rhodopsin misfolding caused by the P23H mutation is a major cause of autosomal dominant retinitis pigmentosa (adRP). To date, there are no effective treatments for adRP. The BiP co-chaperone and reductase ERdj5 (DNAJC10) is part of the endoplasmic reticulum (ER) quality control machinery, and previous studies have shown that overexpression of ERdj5 in vitro enhanced the degradation of P23H rhodopsin, whereas knockdown of ERdj5 increased P23H rhodopsin ER retention and aggregation. Here, we investigated the role of ERdj5 in photoreceptor homeostasis in vivo by using an Erdj5 knockout mouse crossed with the P23H knock-in mouse and by adeno-associated viral (AAV) vector-mediated gene augmentation of ERdj5 in P23H-3 rats. Electroretinogram (ERG) and optical coherence tomography of Erdj5-/- and P23H+/-:Erdj5-/- mice showed no effect of ERdj5 ablation on retinal function or photoreceptor survival. Rhodopsin levels and localization were similar to those of control animals at a range of time points. By contrast, when AAV2/8-ERdj5-HA was subretinally injected into P23H-3 rats, analysis of the full-field ERG suggested that overexpression of ERdj5 reduced visual function loss 10 weeks post-injection (PI). This correlated with a significant preservation of photoreceptor cells at 4 and 10 weeks PI. Assessment of the outer nuclear layer (ONL) morphology showed preserved ONL thickness and reduced rhodopsin retention in the ONL in the injected superior retina. Overall, these data suggest that manipulation of the ER quality control and ER-associated degradation factors to promote mutant protein degradation could be beneficial for the treatment of adRP caused by mutant rhodopsin.

Ablation of the Chaperone Protein ERdj5 Results in a Sjögren's Syndrome-Like Phenotype in Mice, Consistent With an Upregulated Unfolded Protein Response in Human Patients.

Objective: Sjögren's syndrome (SS) is a chronic autoimmune disorder that affects mainly the exocrine glands. Endoplasmic reticulum (ER) stress proteins have been suggested to participate in autoimmune and inflammatory responses, either acting as autoantigens, or by modulating factors of inflammation. The chaperone protein ERdj5 is an ER-resident disulfide reductase, required for the translocation of misfolded proteins during ER-associated protein degradation. In this study we investigated the role of ERdj5 in the salivary glands (SGs), in association with inflammation and autoimmunity. Methods:In situ expression of ERdj5 and XBP1 activation were studied immunohistochemically in minor SG tissues from primary SS patients and non-SS sicca-complaining controls. We used the mouse model of ERdj5 ablation and characterized its features: Histopathological, serological (antinuclear antibodies and cytokine levels), and functional (saliva flow rate). Results: ERdj5 was highly expressed in the minor SGs of SS patients, with stain intensity correlated to inflammatory lesion severity and anti-SSA/Ro positivity. Moreover, SS patients demonstrated higher XBP1 activation within the SGs. Remarkably, ablation of ERdj5 in mice conveyed many of the cardinal features of SS, like spontaneous inflammation in SGs with infiltrating T and B lymphocytes, distinct cytokine signature, excessive cell death, reduced saliva flow, and production of anti-SSA/Ro and anti-SSB/La autoantibodies. Notably, these features were more pronounced in female mice. Conclusions: Our findings suggest a critical connection between the function of the ER chaperone protein ERdj5 and autoimmune inflammatory responses in the SGs and provide evidence for a new, potent animal model of SS.

Implications of the mitochondrial interactome of mammalian thioredoxin 2 for normal cellular function and disease.

Multiple thioredoxin isoforms exist in all living cells. To explore the possible functions of mammalian mitochondrial thioredoxin 2 (Trx2), an interactome of mouse Trx2 was initially created using (i) a monothiol mouse Trx2 species for capturing protein partners from different organs and (ii) yeast two hybrid screens on human liver and rat brain cDNA libraries. The resulting interactome consisted of 195 proteins (Trx2 included) plus the mitochondrial 16S RNA. 48 of these proteins were classified as mitochondrial (MitoCarta2.0 human inventory). In a second step, the mouse interactome was combined with the current four-membered mitochondrial sub-network of human Trx2 (BioGRID) to give a 53-membered human Trx2 mitochondrial interactome (52 interactor proteins plus the mitochondrial 16S RNA). Although thioredoxins are thiol-employing disulfide oxidoreductases, approximately half of the detected interactions were not due to covalent disulfide bonds. This finding reinstates the extended role of thioredoxins as moderators of protein function by specific non-covalent, protein-protein interactions. Analysis of the mitochondrial interactome suggested that human Trx2 was involved potentially in mitochondrial integrity, formation of iron sulfur clusters, detoxification of aldehydes, mitoribosome assembly and protein synthesis, protein folding, ADP ribosylation, amino acid and lipid metabolism, glycolysis, the TCA cycle and the electron transport chain. The oxidoreductase functions of Trx2 were verified by its detected interactions with mitochondrial peroxiredoxins and methionine sulfoxide reductase. Parkinson's disease, triosephosphate isomerase deficiency, combined oxidative phosphorylation deficiency, and lactate dehydrogenase b deficiency are some of the diseases where the proposed mitochondrial network of Trx2 may be implicated.

Lymphocytes eject interferogenic mitochondrial DNA webs in response to CpG and non-CpG oligodeoxynucleotides of class C.

Circulating mitochondrial DNA (mtDNA) is receiving increasing attention as a danger-associated molecular pattern in conditions such as autoimmunity, cancer, and trauma. We report here that human lymphocytes [B cells, T cells, natural killer (NK) cells], monocytes, and neutrophils derived from healthy blood donors, as well as B cells from chronic lymphocytic leukemia patients, rapidly eject mtDNA as web filament structures upon recognition of CpG and non-CpG oligodeoxynucleotides of class C. The release was quenched by ZnCl2, independent of cell death (apoptosis, necrosis, necroptosis, autophagy), and continued in the presence of TLR9 signaling inhibitors. B-cell mtDNA webs were distinct from neutrophil extracellular traps concerning structure, reactive oxygen species (ROS) dependence, and were devoid of antibacterial proteins. mtDNA webs acted as rapid (within minutes) messengers, priming antiviral type I IFN production. In summary, our findings point at a previously unrecognized role for lymphocytes in antimicrobial defense, utilizing mtDNA webs as signals in synergy with cytokines and natural antibodies, and cast light on the interplay between mitochondria and the immune system.

The expression and activity of thioredoxin reductase 1 splice variants v1 and v2 regulate the expression of genes associated with differentiation and adhesion.

The mammalian redox-active selenoprotein thioredoxin reductase (TrxR1) is a main player in redox homoeostasis. It transfers electrons from NADPH to a large variety of substrates, particularly to those containing redox-active cysteines. Previously, we reported that the classical form of cytosolic TrxR1 (TXNRD1_v1), when overexpressed in human embryonic kidney cells (HEK-293), prompted the cells to undergo differentiation [Nalvarte et al. (2004) J. Biol. Chem. 279: , 54510-54517]. In the present study, we show that several genes associated with differentiation and adhesion are differentially expressed in HEK-293 cells stably overexpressing TXNRD1_v1 compared with cells expressing its splice variant TXNRD1_v2. Overexpression of these two splice forms resulted in distinctive effects on various aspects of cellular functions including gene regulation patterns, alteration of growth rate, migration and morphology and susceptibility to selenium-induced toxicity. Furthermore, differentiation of the neuroblastoma cell line SH-SY5Y induced by all-trans retinoic acid (ATRA) increased both TXNRD1_v1 and TXNRD1_v2 expressions along with several of the identified genes associated with differentiation and adhesion. Selenium supplementation in the SH-SY5Y cells also induced a differentiated morphology and changed expression of the adhesion protein fibronectin 1 and the differentiation marker cadherin 11, as well as different temporal expression of the studied TXNRD1 variants. These data suggest that both TXNRD1_v1 and TXNRD1_v2 have distinct roles in differentiation, possibly by altering the expression of the genes associated with differentiation, and further emphasize the importance in distinguishing each unique action of different TrxR1 splice forms, especially when studying the gene silencing or knockout of TrxR1.

Glutaredoxin-1 regulates TRAF6 activation and the IL-1 receptor/TLR4 signalling.

Glutaredoxin-1 (GRX-1) is a cytoplasmic enzyme that highly contributes to the antioxidant defense system. It catalyzes the reversible reduction of glutathione-protein mixed disulfides, a process called deglutathionylation. Here, we investigated the role of GRX-1 in the pathway triggered by interleukin-1/Toll-like receptor 4 (IL-1R/TLR4) by using RNA interference (RNAi) in HEK293 and HeLa cells. TNF receptor-associated factor 6 (TRAF6) is an intermediate signalling molecule involved in the signal transduction by members of the interleukin-1/Toll-like receptor (IL-1R/TLR) family. TRAF6 has an E3 ubiquitin ligase activity which depends on the integrity of an amino-terminal really interesting new gene (RING) finger motif. Upon receptor activation, TRAF6 undergoes K63-linked auto-polyubiquitination which mediates protein-protein interactions and signal propagation. Our data showed that IL-1R and TLR4-mediated NF-κB induction was severely reduced in GRX-1 knockdown cells. We found that the RING-finger motif of TRAF6 is S-glutathionylated under normal conditions. Moreover, upon IL-1 stimulation TRAF6 undergoes deglutathionylation catalyzed by GRX-1. The deglutathionylation of TRAF6 is essential for its auto-polyubiquitination and subsequent activation. Taken together, our findings reveal another signalling molecule affected by S-glutathionylation and uncover a crucial role for GRX-1 in the TRAF6-dependent activation of NF-κB by IL-1R/TLRs.

Interaction of mitochondrial thioredoxin with glucocorticoid receptor and NF-kappaB modulates glucocorticoid receptor and NF-kappaB signalling in HEK-293 cells.

Trx2 (mitochondrial thioredoxin) is an antioxidant and anti-apoptotic factor essential for cell viability. Trx1 (cytoplasmic thioredoxin) is a co-factor and regulator of redox-sensitive transcription factors such as the GR (glucocorticoid receptor) and NF-kappaB (nuclear factor kappaB). Both transcription factors have been detected in mitochondria and a role in mitochondrial transcription regulation and apoptosis has been proposed. In the present study, we show using SPR (surface plasmon resonance) and immunoprecepitation that GR and the p65 subunit of NF-kappaB are Trx2-interacting proteins. The interaction of Trx2 with GR is independent of the presence of GR ligand and of redox conditions. The p65 subunit of NF-kappaB can interact with Trx2 in the oxidized, but not the reduced, form. Using HEK (human embryonic kidney)-293 cell lines with increased or decreased expression of Trx2, we show that Trx2 modulates transcription of GR and NF-kappaB reporter genes. Moreover, Trx2 overexpression modulates the mRNA levels of the COX1 (cytochrome oxidase subunit I) and Cytb (cytochrome b), which are known to be regulated by GR and NF-kappaB. Increased expression of Trx2 differentially affects the expression of Cytb. The glucocorticoid dexamethasone potentiates the expression of Cytb, whereas TNFalpha (tumour necrosis factor alpha) down-regulates it. These results suggest a regulatory role for Trx2 in GR and NF-kappaB signalling pathways.

ERdj5 sensitizes neuroblastoma cells to endoplasmic reticulum stress-induced apoptosis.

Down-regulation of the unfolded protein response (UPR) can be therapeutically valuable in cancer treatment, and endoplasmic reticulum (ER)-resident chaperone proteins may thus be targets for developing novel chemotherapeutic strategies. ERdj5 is a novel ER chaperone that regulates the ER-associated degradation of misfolded proteins through its associations with EDEM and the ER stress sensor BiP. To investigate whether ERdj5 can regulate ER stress signaling pathways, we exposed neuroblastoma cells overexpressing ERdj5 to ER stress inducers. ERdj5 promoted apoptosis in tunicamycin, thapsigargin, and bortezomib-treated cells. To provide further evidence that ERdj5 induces ER stress-regulated apoptosis, we targeted Bcl-2 to ER of ERdj5-overexpressing cells. Targeting the Bcl-2 to ER prevented the apoptosis induced by ER stress inducers but not by non-ER stress apoptotic stimuli, suggesting induction of ER stress-regulated apoptosis by ERdj5. ERdj5 enhanced apoptosis by abolishing the ER stress-induced phosphorylation of eukaryotic translation initiation factor 2alpha (eIF2alpha) and the subsequent translational repression. ERdj5 was found to inhibit the eIF2alpha phosphorylation under ER stress through inactivating the pancreatic endoplasmic reticulum kinase. The compromised integrated stress response observed in ERdj5-overexpressing ER-stressed cells due to repressed eIF2alpha phosphorylation correlated with impaired neuroblastoma cell resistance under ER stress. These results demonstrate that ERdj5 decreases neuroblastoma cell survival by down-regulating the UPR, raising the possibility that this protein could be a target for anti-tumor approaches.

S-glutathionylation of IRF3 regulates IRF3-CBP interaction and activation of the IFN beta pathway.

Interferon regulatory factor 3 (IRF3) is an essential transcriptional regulator of the interferon genes. IRF3 is constitutively present in a latent conformation in the cell cytoplasm. In cells infected by Sendai virus, IRF3 becomes phosphorylated, homodimerizes, translocates to the nucleus, binds to target genes and activates transcription by interacting with CBP/p300 co-activators. In this study, we report that in non-infected cells IRF3 is post-translationally modified by S-glutathionylation. Upon viral-infection, it undergoes a deglutathionylation step that is controlled by the cytoplasmic enzyme glutaredoxin-1 (GRX-1). In virus-infected GRX-1 knockdown cells, phosphorylation, homodimerization and nuclear translocation of IRF3 were not affected, but the transcriptional activity of IRF3 and the expression of interferon-beta (IFNbeta), were severely reduced. We show that deglutathionylation of IRF3 is necessary for efficient interaction of IRF3 with CBP, an event essential for transcriptional activation of the interferon genes. Taken together, these findings reveal a crucial role for S-glutathionylation and GRX-1 in controlling the activation of IRF3 and IFNbeta gene expression.

The mitochondrial thioredoxin system regulates nitric oxide-induced HIF-1alpha protein.

Hypoxia-inducible factor-1 (HIF-1), consisting of two subunits, HIF-1alpha and HIF-1beta, is a key regulator for adaptation to low oxygen availability, i.e., hypoxia. Compared to the constitutively expressed HIF-1beta, HIF-1alpha is regulated by hypoxia but also under normoxia (21% O(2)) by several stimuli, including nitric oxide (NO). In this study, we present evidence that overexpression of mitochondrial-located thioredoxin 2 (Trx2) or thioredoxin reductase 2 (TrxR2) attenuated NO-evoked HIF-1alpha accumulation and transactivation of HIF-1 in HEK293 cells. In contrast, cytosolic-located thioredoxin 1 (Trx1) enhanced HIF-1alpha protein amount and activity under NO treatments. Taking into consideration that thioredoxins affect the synthesis of HIF-1alpha by altering Akt/mTOR signaling, we herein show that p42/44 mitogen-activated protein kinase and p70S6 kinase are involved. Moreover, intracellular ATP was increased in Trx1-overexpressing cells but reduced in cells overexpressing Trx2 or TrxR2, providing thus an understanding of how protein synthesis is regulated by thioredoxins.

Ligands differentially modify the nuclear mobility of estrogen receptors alpha and beta.

Signaling of nuclear receptors depends on the structure of their ligands, with different ligands eliciting different responses. In this study using a comparative analysis, an array of ligands was examined for effects on estrogen receptor alpha (ERalpha) and ERbeta mobility. Our results indicated that these two receptors share similarities in response to some ligands but differ significantly in response to others. Our results suggest that for ERalpha, ligands can be classified into three distinct groups: 1) ligands that do not affect the mobility of the receptor, 2) ligands that cause a moderate effect, and 3) ligands that strongly impact mobility of ERalpha. Interestingly, we found that for ERbeta such a classification was not possible because ERbeta ligands caused a wider spectrum of responses. One of the main differences between the two receptors was the response toward the antiestrogens ICI and raloxifene, which was not attributable to differential subnuclear localization or different conformations of helix 12 in the C-terminal domain. We showed that both of these ligands caused a robust phenotype, leading to an almost total immobilization of ERalpha, whereas ERbeta retained its mobility; we provide evidence that the mobility of the two receptors depends upon the function of the proteasome machinery. This novel finding that ERbeta retains its mobility in the presence of antiestrogens could be important for its ability to regulate genes that do not contain classic estrogen response element sites and do not require DNA binding and could be used in the investigation of ligands that show ER subtype specificity.

Overexpression of thioredoxin reductase 1 inhibits migration of HEK-293 cells.

BACKGROUND INFORMATION: TrxR (thioredoxin reductase), in addition to protecting against oxidative stress, plays a role in the redox regulation of intracellular signalling pathways controlling, among others, cell proliferation and apoptosis. The aim of the present study was to determine whether TrxR1 is involved in the regulation of cell migration. RESULTS: Stably transfected HEK-293 (human embryonic kidney) cells which overexpress cytosolic TrxR1 (HEK-TrxR15 and HEK-TrxR11 cells) were used in the present study. We found that the stimulation of cell motility induced by PKC (protein kinase C) activators, PMA and DPhT (diphenyltin), was inhibited significantly in the HEK-TrxR15 and HEK-TrxR11 cells compared with control cells. The overexpression of TrxR1 also inhibited characteristic morphological changes and reorganization of the F-actin cytoskeleton induced by PMA and DPhT. In addition, the selective activation of PKCdelta by DPhT was inhibited in cells that overexpressed cytosolic TrxR1. Furthermore, rottlerin, a selective inhibitor of PKCdelta, and PKCdelta siRNA (small interfering RNA), suppressed the morphological changes induced by DPhT in the control cells. CONCLUSIONS: The overexpression of TrxR1 inhibits migration of HEK-293 cells stimulated with PMA and DPhT. Moreover, our observations suggest that this effect is mediated by the inhibition of PKCdelta activation.

Nuclear immobilization of DsRed1 tagged proteins: a novel tool for studying DNA-protein interactions?

DsRed1 is a red fluorescent protein that can be used as a fusion partner with other proteins to determine their subcellular localization, similarly to the popular green fluorescent proteins (GFP). Here, we report that fusion of DsRed1 to estrogen receptor alpha (ER alpha) renders the transcription factor immobile within the nucleus. Furthermore, we show that the immobilization is dependent on DNA interaction and that the binding to the DNA can be direct as well as indirect for DsRed to immobilize with its fusion partners. This observation could provide a new tool to be used for the identification of target genes containing low affinity binding sites for several transcription factors including ER alpha. In addition, it could be employed for studies on protein-DNA interactions as well as protein-protein interactions during protein complex formation on chromatin in the event of transcription initiation and regulation.

Thioredoxin 1 and thioredoxin 2 have opposed regulatory functions on hypoxia-inducible factor-1alpha.

Hypoxia inducible factor 1 (HIF-1), a key regulator for adaptation to hypoxia, is composed of HIF-1alpha and HIF-1beta. In this study, we present evidence that overexpression of mitochondria-located thioredoxin 2 (Trx2) attenuated hypoxia-evoked HIF-1alpha accumulation, whereas cytosolic thioredoxin 1 (Trx1) enhanced HIF-1alpha protein amount. Transactivation of HIF-1 is decreased by overexpression of Trx2 but stimulated by Trx1. Inhibition of proteasomal degradation of HIF-1alpha in Trx2-overexpressing cells did not fully restore HIF-1alpha protein levels, while HIF-1alpha accumulation was enhanced in Trx1-overexpressing cells. Reporter assays showed that cap-dependent translation is increased by Trx1 and decreased by Trx2, whereas HIF-1alpha mRNA levels remained unaltered. These data suggest that thioredoxins affect the synthesis of HIF-1alpha. Trx1 facilitated synthesis of HIF-1alpha by activating Akt, p70S6K, and eIF-4E, known to control cap-dependent translation. In contrast, Trx2 attenuated activities of Akt, p70S6K, and eIF-4E and provoked an increase in mitochondrial reactive oxygen species production. MitoQ, a mitochondria specific antioxidant, reversed HIF-1alpha accumulation as well as Akt activation under hypoxia in Trx2 cells, supporting the notion of translation control mechanisms in affecting HIF-1alpha protein accumulation.

Glucocorticoid receptor isoforms in human hepatocarcinoma HepG2 and SaOS-2 osteosarcoma cells: presence of glucocorticoid receptor alpha in mitochondria and of glucocorticoid receptor beta in nucleoli.

In the context of a possible direct action of glucocorticosteroids on mitochondrial transcription and/or apoptosis by way of cognate mitochondrial receptors, the possible localization of glucocorticoid receptors alpha and beta (GRalpha and GRbeta) in mitochondria was explored in human hepatocarcinoma HepG2 and osteosarcoma SaOS-2 cells, in which glucocorticoids exert an anabolic and apoptotic effect, respectively. In both cell types, GRalpha was detected in mitochondria, in nuclei and in cytosol by immunofluorescence labeling and confocal scanning microscopy, by immunogold electron microscopy and by Western blotting. GRbeta was shown to be almost exclusively restricted to the nucleus of the two cell types, being particularly concentrated in nucleoli, pointing to a solely nuclear role of this receptor isoform and to a possible function in nucleoli related processes. Computer analysis identified a putative internal mitochondrial targeting sequence within the glucocorticoid receptor. The demonstration of mitochondrially localized GRalpha in HepG2 and SaOS-2 cells corroborates previous findings in other cell types and further supports a direct role of this receptor in mitochondrial functions.

The role of thioredoxin reductase activity in selenium-induced cytotoxicity.

The selenoprotein thioredoxin reductase is a key enzyme in selenium metabolism, reducing selenium compounds and thereby providing selenide to synthesis of all selenoproteins. We evaluated the importance of active TrxR1 in selenium-induced cytotoxicity using transfected TrxR1 over-expressing stable Human Embryo Kidney (HEK-293) cells and modulation of activity by pretreatment with low concentration of selenite. Treatment with sodium selenite induced cytotoxity in a dose-dependent manner in both TrxR1 over-expressing and control cells. However, TrxR1 over-expressing cells, which were preincubated for 72h with 0.1 microM selenite, were significantly more resistant to selenite cytotoxicity than control cells. To demonstrate the early effects of selenite on behaviour of HEK-293 cells, we also investigated the influence of this compound on cell motility. We observed inhibition of cell motility by 50 microM selenite immediately after administration. Moreover, TrxR1 over-expressing cells preincubated with a low concentration of selenite were more resistant to the inhibitory effect of 50 microM selenite than those not preincubated. It was also observed that the TrxR over-expressing cells showed higher TrxR1 activity than control cells and the preincubation of over-expressing cells with 0.1 microM selenite induced further significant increase in the activity of TrxR1. On the other hand, we demonstrated that TrxR1 over-expressing cells showed decreased glutathione peroxidase activity compared to control cells. These data strongly suggest that TrxR1 may be a crucial enzyme responsible for cell resistance against selenium cytotoxicity.

Overexpression of enzymatically active human cytosolic and mitochondrial thioredoxin reductase in HEK-293 cells. Effect on cell growth and differentiation.

The mammalian thioredoxin reductases (TrxR) are selenoproteins containing a catalytically active selenocysteine residue (Sec) and are important enzymes in cellular redox control. The cotranslational incorporation of Sec, necessary for activity, is governed by a stem-loop structure in the 3'-untranslated region of the mRNA and demands adequate selenium availability. The complicated translation machinery required for Sec incorporation is a major obstacle in isolating mammalian cell lines stably overexpressing selenoproteins. In this work we report on the development and characterization of stably transfected human embryonic kidney 293 cells that overexpress enzymatically active selenocysteine-containing cytosolic TrxR1 or mitochondrial TrxR2. We demonstrate that the overexpression of selenium-containing TrxR1 results in lower expression and activity of the endogenous selenoprotein glutathione peroxidase and that the activity of overexpressed TrxRs, rather than the protein amount, can be increased by selenium supplementation in the cell growth media. We also found that the TrxR-overexpressing cells grew slower over a wide range of selenium concentrations, which was an effect apparently not related to increased apoptosis nor to fatally altered intracellular levels of reactive oxygen species. Most surprisingly, the TrxR1- or TrxR2-overexpressing cells also induced novel expression of the epithelial markers CK18, CK-Cam5.2, and BerEP4, suggestive of a stimulation of cellular differentiation.