Transcript and Protein Analysis Reveals Better Survival Skills of Monocyte-Derived Dendritic Cells Compared to Monocytes during Oxidative Stress

Background

Dendritic cells (DCs), professional antigen-presenting cells with the unique ability to initiate primary T-cell responses, are present in atherosclerotic lesions where they are exposed to oxidative stress that generates cytotoxic reactive oxygen species (ROS). A large body of evidence indicates that cell death is a major modulating factor of atherogenesis. We examined antioxidant defence systems of human monocyte-derived (mo)DCs and monocytes in response to oxidative stress.

Methods

Oxidative stress was induced by addition of tertiary-butylhydroperoxide (tert-BHP, 30 min). Cellular responses were evaluated using flow cytometry and confocal live cell imaging (both using 5-(and-6)-chloromethyl-2,7-dichlorodihydrofluorescein diacetate, CM-H₂DCFDA). Viability was assessed by the neutral red assay. Total RNA was extracted for a PCR profiler array. Five genes were selected for confirmation by Taqman gene expression assays, and by immunoblotting or immunohistochemistry for protein levels.

Results

Tert-BHP increased CM-H₂DCFDA fluorescence and caused cell death. Interestingly, all processes occurred more slowly in moDCs than in monocytes. The mRNA profiler array showed more than 2-fold differential expression of 32 oxidative stress–related genes in unstimulated moDCs, including peroxiredoxin-2 (PRDX2), an enzyme reducing hydrogen peroxide and lipid peroxides. PRDX2 upregulation was confirmed by Taqman assays, immunoblotting and immunohistochemistry. Silencing PRDX2 in moDCs by means of siRNA significantly increased CM-DCF fluorescence and cell death upon tert-BHP-stimulation.

Conclusions

Our results indicate that moDCs exhibit higher intracellular antioxidant capacities, making them better equipped to resist oxidative stress than monocytes. Upregulation of PRDX2 is involved in the neutralization of ROS in moDCs. Taken together, this points to better survival skills of DCs in oxidative stress environments, such as atherosclerotic plaques.     

Proteomics analysis identifies PARK7 as an important player for renal cell resistance and survival under oxidative stress

Renal fibrosis is a process that is characterized by declining excretory renal function. The molecular mechanisms of fibrosis are not fully understood. Oxidative stress pathways were reported to be involved in renal tissue deterioration and fibrosis progression. In order to identify new molecular targets associated with oxidative stress and renal fibrosis, differential proteomics analysis was performed with established renal cell lines (TK173 and HK-2). The cells were treated with oxidative stress triggering factor H(2)O(2) and the proteome alterations were investigated. Two dimensional protein maps were generated and differentially expressed proteins were processed and identified using mass spectrometry analysis combined with data base search. Interestingly the increase of ROS in the renal cell lines upon H(2)O(2) treatment was accompanied by alteration of a large number of proteins, which could be classified in three categories: the first category grouped the proteins that have been described to be involved in fibrogenesis (e.g. ACTA2, VIN, VIM, DES, KRT, COL1A1, COL4A1), the second category, which was more interesting involved proteins of the oxidative stress pathway (PRDX1, PRDX2, PRDX6, SOD, PARK7, HYOU1), which were highly up-regulated under oxidative stress, and the third category represented proteins, which are involved in different other metabolic pathways. Among the oxidative stress proteins the up-regulation of PARK7 was accompanied by a shift in the pI as a result of oxidation. Knockdown of PARK7 using siRNA led to significant reduction in renal cell viability under oxidative stress. Under H(2)O(2) treatment the PARK7 knockdown cells showed up to 80% decrease in cell viability and an increase in apoptosis compared to the controls. These results highlight for the first time the important role of PARK7 in oxidative stress resistance in renal cells.     

Characterization and expression of Peroxiredoxin 1 in the neonatal tammar wallaby (Macropus eugenii)

Peroxiredoxin 1 (PRDX1) is a ubiquitously expressed antioxidant with vital roles in basal metabolic functions. In addition PRDX1 is involved in cell differentiation and proliferation, apoptosis and innate immunity. In this study, we have characterized PRDX1 from the tammar wallaby (Macropus eugenii). Tammar PRDX1 has high conservation of functional residues and motifs, and demonstrates a close homology with eutherian and vertebrate orthologues. Stimulation of adult tammar leukocytes with lipopolysaccharide and lipoteichoic acid suggests a role for PRDX1 in innate immune defences. PRDX1 expression in the organs of tammar pouch young was mildly elevated early in life possibly reflecting its role in basal metabolic processes. Later increases in PRDX1 expression correlated with functional maturation of several immune organs or with preparation for increased oxidative stress of emergence. The findings of the study are reflections of the complex integrated roles that PRDX1 has in regulation of oxidative stress, apoptosis, cell differentiation and proliferation, and innate immunity.     

Antioxidant enzyme peroxiredoxin 5 is upregulated in degenerative human tendon

Peroxiredoxin 5 (PRDX5) is a novel thioredoxin peroxidase recently identified in a variety of human cells and tissues, which is considered to play an important role in oxidative stress protection mechanisms. However, little is known about its expression in tendon degeneration, a common and disabling condition that primarily affects older people, in which oxidative stress may be implicated. The present study demonstrated that normal human tendon expresses PRDX5 and its expression is significantly increased in degenerative tendon. In addition, we have localized PRDX5 to fibroblasts in normal tendon and to both fibroblasts and endothelial cells in degenerate tendon. The differential expression of PRDX5 in normal and degenerate tendon shows that a thioredoxin peroxidase with antioxidant properties is upregulated under pathophysiological conditions and suggests that oxidative stress may be involved in the pathogenesis of tendon degeneration. PRDX5 may play a protective role against oxidative stress during this pathophysiological process.     

Vitamin K epoxide reductase contributes to protein disulfide formation and redox homeostasis within the endoplasmic reticulum

The transfer of oxidizing equivalents from the endoplasmic reticulum (ER) oxidoreductin (Ero1) oxidase to protein disulfide isomerase is an important pathway leading to disulfide formation in nascent proteins within the ER. However, Ero1-deficient mouse cells still support oxidative protein folding, which led to the discovery that peroxiredoxin IV (PRDX4) catalyzes a parallel oxidation pathway. To identify additional pathways, we used RNA interference in human hepatoma cells and evaluated the relative contributions to oxidative protein folding and ER redox homeostasis of Ero1, PRDX4, and the candidate oxidants quiescin-sulfhydryl oxidase 1 (QSOX1) and vitamin K epoxide reductase (VKOR). We show that Ero1 is primarily responsible for maintaining cell growth, protein secretion, and recovery from a reductive challenge. We further show by combined depletion with Ero1 that PRDX4 and, for the first time, VKOR contribute to ER oxidation and that depletion of all three activities results in cell death. Of importance, Ero1, PRDX4, or VKOR was individually capable of supporting cell viability, secretion, and recovery after reductive challenge in the near absence of the other two activities. In contrast, no involvement of QSOX1 in ER oxidative processes could be detected. These findings establish VKOR as a significant contributor to disulfide bond formation within the ER.     

Downregulated Expression of Peroxiredoxin 4 in Granulosa Cells from Polycystic Ovary Syndrome

Peroxiredoxin 4 (PRDX4), a member of Peroxiredoxin (PRDX) family, is a typical 2-Cys PRDX. PRDX4 monitors the oxidative burden within cellular compartment and reduces hydrogen peroxide and alkyl hydroperoxide related to oxidative stress and apoptosis. Antioxidant, like PRDX4, may promote follicle development and participate in the pathophysiology of PCOS. In our previous study, we found that PRDX4 was expressed in mice oocyte cumulus oophorus complex, and that PRDX4 could be associated with follicle development. In this study, we explored the expression of PRDX4 in human follicles and possible role of PRDX4 in PCOS pathophysiology. Our data showed that PRDX4 was mainly expressed in granulosa cells in human ovaries. When compared to control group, both PRDX4 mRNA level and protein level decreased in PCOS group. The lowered levels of PRDX4 may relate to oxidative stress in the pathophysiologic progress of PCOS. Furthermore, expression of PRDX4 in the granulosa cells of in vivo or in vitro matured follicles was higher than that in immatured follicles, which suggested that PRDX4 may have a close relationship with follicular development. Altogether, our findings may provide new clues of the pathophysiologic mechanism of PCOS and potential therapeutic strategy using antioxidant, like PRDX4.     

Ferroptosis-inducing agents enhance TRAIL-induced apoptosis through upregulation of death receptor 5

Ferroptosis is considered genetically and biochemically distinct from other forms of cell death. In this study, we examined whether ferroptosis shares cell death pathways with other types of cell death. When human colon cancer HCT116, CX-1, and LS174T cells were treated with ferroptotic agents such as sorafenib (SRF), erastin, and artesunate, data from immunoblot assay showed that ferroptotic agents induced endoplasmic reticulum (ER) stress and the ER stress response-mediated expression of death receptor 5 (DR5), but not death receptor 4. An increase in the level of DR5, which is activated by binding to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and initiates apoptosis, was probably responsible for synergistic apoptosis when cells were treated with ferroptotic agent in combination with TRAIL. This collateral effect was suppressed in C/EBP (CCAAT-enhancer-binding protein)-homologous protein (CHOP)-deficient mouse embryonic fibroblasts or DR5 knockdown HCT116 cells, but not in p53-deficient HCT116 cells. The results from in vitro studies suggest the involvement of the p53-independent CHOP/DR5 axis in the synergistic apoptosis during the combinatorial treatment of ferroptotic agent and TRAIL. The synergistic apoptosis and regression of tumor growth were also observed in xenograft tumors when SRF and TRAIL were administered to tumor-bearing mice.     

Role of peroxiredoxin-2 in protecting RBCs from hydrogen peroxide-induced oxidative stress

Abstract

The role of peroxiredoxin-2 (PRDX2) in preventing hydrogen peroxide-induced oxidative stress in the red blood cell was investigated by comparing blood from PRDX2 knockout mice with superoxide dismutase-1 (SOD1) knockout and control mice. Loss of PRDX2 increased basal levels of methemoglobin and heme degradation (a marker for oxidative stress), and reduced red blood cell deformability. In vitro incubation under normoxic conditions, both with and without inhibition of catalase, resulted in a lag phase during which negligible heme degradation occurred followed by a more rapid rate of heme degradation in the absence of PRDX2. The appreciable basal increase in heme degradation for PRDX2 knockout mice, together with the lag during in vitro incubation, implies that PRDX2 neutralizes hydrogen peroxide generated in vivo under the transient hypoxic conditions experienced as the cells pass through the microcirculation.     

TRAIL-Based High Throughput Screening Reveals a Link between TRAIL-Mediated Apoptosis and Glutathione Reductase,a Key Component of Oxidative Stress Response

A high throughput screen for compounds that induce TRAIL-mediated apoptosis identified ML100 as an active chemical probe, which potentiated TRAIL activity in prostate carcinoma PPC-1 and melanoma MDA-MB-435 cells. Follow-up in silico modeling and profiling in cell-based assays allowed us to identify NSC130362, pharmacophore analog of ML100 that induced 65-95% cytotoxicity in cancer cells and did not affect the viability of human primary hepatocytes. In agreement with the activation of the apoptotic pathway, both ML100 and NSC130362 synergistically with TRAIL induced caspase-3/7 activity in MDA-MB-435 cells. Subsequent affinity chromatography and inhibition studies convincingly demonstrated that glutathione reductase (GSR), a key component of the oxidative stress response, is a target of NSC130362. In accordance with the role of GSR in the TRAIL pathway, GSR gene silencing potentiated TRAIL activity in MDA-MB-435 cells but not in human hepatocytes. Inhibition of GSR activity resulted in the induction of oxidative stress, as was evidenced by an increase in intracellular reactive oxygen species (ROS) and peroxidation of mitochondrial membrane after NSC130362 treatment in MDA-MB-435 cells but not in human hepatocytes. The antioxidant reduced glutathione (GSH) fully protected MDA-MB-435 cells from cell lysis induced by NSC130362 and TRAIL, thereby further confirming the interplay between GSR and TRAIL. As a consequence of activation of oxidative stress, combined treatment of different oxidative stress inducers and NSC130362 promoted cell death in a variety of cancer cells but not in hepatocytes in cell-based assays and in in vivo, in a mouse tumor xenograft model.     

Three-dimensional microscopy characterization of death receptor 5 expression by over-activated human primary CD4+ T cells and apoptosis

Activation-induced cell death is a natural process that prevents tissue damages from over-activated immune cells. TNF-Related apoptosis ligand (TRAIL), a TNF family member, induces apoptosis of infected and tumor cells by binding to one of its two death receptors, DR4 or DR5. TRAIL was reported to be secreted by phytohemagglutinin (PHA)-stimulated CD4(+) T cells in microvesicles.We investigate here TRAIL and DR5 regulation by activated primary CD4(+) T cells and its consequence on cell death. We observed that PHA induced CD4(+) T cell apoptosis in a dose-dependent manner. Thus, we investigated molecules involved in PHA-mediated cell death and demonstrated that TRAIL and DR5 were over-expressed on the plasma membrane of PHA-stimulated CD4(+) T cells. Surprisingly, DR5 was constitutively expressed in naive CD4(+) T cells at messenger RNA (mRNA) and protein levels. Thus, using 3 dimensional microscopy and intracellular staining assays, we show that DR5 is constitutively expressed in CD4(+) T cells and is pre-stocked in the cytoplasm. When cells are stimulated by PHA, DR5 is relocalized from cytoplasm to plasma membrane. Small interference RNA (siRNA) and blocking antibody assays demonstrate that TRAIL/DR5 interaction is mainly responsible for PHA-mediated CD4(+) T cell apoptosis. Thus, membrane DR5 expression leading to TRAIL-mediated apoptosis may represent one of the pathways responsible for eradication of over-activated CD4(+) T cells during immune responses.     

Tributyltin induces oxidative stress and neuronal injury by inhibiting glutathione S-transferase in rat organotypic hippocampal slice cultures

Tributyltin (TBT) has been used as a heat stabilizer, agricultural pesticide and antifouling agents on ships, boats and fish-farming nets; however, the neurotoxicity of TBT has recently become a concern. TBT is suggested to stimulate the generation of reactive oxygen species (ROS) inside cells. The aim of this study was to determine the mechanism of neuronal oxidative injury induced by TBT using rat organotypic hippocampal slice cultures. The treatment of rat hippocampal slices with TBT induced ROS production, lipid peroxidation and cell death. Pretreatment with antioxidants such as superoxide dismutase, catalase or trolox, suppressed the above phenomena induced by TBT, indicating that TBT elicits oxidative stress in hippocampal slices, which causes neuronal cell death. TBT dose-dependently inhibited glutathione S-transferase (GST), but not glutathione peroxidase or glutathione reductase in the cytosol of rat hippocampus. The treatment of hippocampal slices with TBT decreased the GST activity. Pretreatment with reduced glutathione attenuated the reduction of GST activity and cell death induced by TBT, indicating that the decrease in GST activity by TBT is involved in hippocampal cell death. When hippocampal slices were treated with sulforaphane, the expression and activity of GST were increased. Notably, TBT-induced oxidative stress and cell death were significantly suppressed by pretreatment with sulforaphane. These results indicate that GST inhibition could contribute, at least in part, to the neuronal cell death induced by TBT in hippocampal slices. This study is the first report to show the link between neuronal oxidative injury and the GST inhibition elicited by TBT.