Use of proteomics to demonstrate a hierarchical oxidative stress response to diesel exhaust particle chemicals in a macrophage cell line

Epidemiological studies demonstrate an association between short term exposure to ambient particulate matter (PM) and cardiorespiratory morbidity and mortality. Although the biological mechanisms of these adverse effects are unknown, emerging data suggest a key role for oxidative stress. Ambient PM and diesel exhaust particles (DEP) contain redox cycling organic chemicals that induce pro-oxidative and pro-inflammatory effects in the lung. These responses are suppressed by N-acetylcysteine (NAC), which directly complexes to electrophilic DEP chemicals and exert additional antioxidant effects at the cellular level. A proteomics approach was used to study DEP-induced responses in the macrophage cell line, RAW 264.7. We demonstrate that in the dose range 10-100 microg/ml, organic DEP extracts induce a progressive decline in the cellular GSH/GSSG ratio, in parallel with a linear increase in newly expressed proteins on the two-dimensional gel. Using matrix-assisted laser desorption ionization time-of-flight mass spectrometry and electrospray ionization-liquid chromatography/mass spectrometry/mass spectrometry analysis, 32 newly induced/NAC-suppressed proteins were identified. These include antioxidant enzymes (e.g. heme oxygenase-1 and catalase), pro-inflammatory components (e.g. p38MAPK and Rel A), and products of intermediary metabolism that are regulated by oxidative stress. Heme oxygenase-1 was induced at low extract dose and with minimal decline in the GSH/GSSG ratio, whereas MAP kinase activation required a higher chemical dose and incremental levels of oxidative stress. Moreover, at extract doses >50 microg/ml, there is a steep decline in cellular viability. These data suggest that DEP induce a hierarchical oxidative stress response in which some of these proteins may serve as markers for oxidative stress during PM exposures.     

Comparison of the pro-oxidative and proinflammatory effects of organic diesel exhaust particle chemicals in bronchial epithelial cells and macrophages

Inhaled diesel exhaust particles (DEP) exert proinflammatory effects in the respiratory tract. This effect is related to the particle content of redox cycling chemicals and is involved in the adjuvant effects of DEP in atopic sensitization. We demonstrate that organic chemicals extracted from DEP induce oxidative stress in normal and transformed bronchial epithelial cells, leading to the expression of heme oxygenase 1, activation of the c-Jun N-terminal kinase cascade, IL-8 production, as well as induction of cytotoxicity. Among these effects, heme oxygenase 1 expression is the most sensitive marker for oxidative stress, while c-Jun N-terminal kinase activation and induction of apoptosis-necrosis require incremental amounts of the organic chemicals and increased levels of oxidative stress. While a macrophage cell line (THP-1) responded in similar fashion, epithelial cells produced more superoxide radicals and were more susceptible to cytotoxic effects than macrophages. Cytotoxicity is the result of mitochondrial damage, which manifests as ultramicroscopic changes in organelle morphology, a decrease in the mitochondrial membrane potential, superoxide production, and ATP depletion. Epithelial cells also differ from macrophages in not being protected by a thiol antioxidant, N-acetylcysteine, which effectively protects macrophages against cytotoxic DEP chemicals. These findings show that epithelial cells exhibit a hierarchical oxidative stress response that differs from that of macrophages by more rapid transition from cytoprotective to cytotoxic responses. Moreover, epithelial cells are not able to convert N-acetylcysteine to cytoprotective glutathione.     

HSP105 interacts with GRP78 and GSK3 and promotes ER stress-induced caspase-3 activation

Stress of the endoplasmic reticulum (ER stress) is caused by the accumulation of misfolded proteins, which occurs in many neurodegenerative diseases. ER stress can lead to adaptive responses or apoptosis, both of which follow activation of the unfolded protein response (UPR). Heat shock proteins (HSP) support the folding and function of many proteins, and are important components of the ER stress response, but little is known about the role of one of the major large HSPs, HSP105. We identified several new partners of HSP105, including glycogen synthase kinase-3 (GSK3), a promoter of ER stress-induced apoptosis, and GRP78, a key component of the UPR. Knockdown of HSP105 did not alter UPR signaling after ER stress, but blocked caspase-3 activation after ER stress. In contrast, caspase-3 activation induced by genotoxic stress was unaffected by knockdown of HSP105, suggesting ER stress-specificity in the apoptotic action of HSP105. However, knockdown of HSP105 did not alter cell survival after ER stress, but instead diverted signaling to a caspase-3-independent cell death pathway, indicating that HSP105 is necessary for apoptotic signaling after UPR activation by ER stress. Thus, HSP105 appears to chaperone the responses to ER stress through its interactions with GRP78 and GSK3, and without HSP105 cell death following ER stress proceeds by a non-caspase-3-dependent process.     

Divergent stress responses to IL-1beta, nitric oxide, and tunicamycin by chondrocytes

As the only cell in cartilage responsible for matrix synthesis, the chondrocyte's viability is crucial to healthy tissue. It must tolerate stresses from both mechanical and cellular sources. This study examines the endoplasmic reticulum (ER) stress response in chondrocytes after exposure to IL-1beta, nitric oxide, or tunicamycin in order to determine whether this form of stress causes cell death. Cultures of the immortalized human juvenile costal chondrocyte cell line, C-28/I2, were treated with IL-1beta, S-nitroso-N-acetylpenicillamine (SNAP), and tunicamycin. Increasing intracellular nitric oxide levels by SNAP treatment or inhibiting protein folding in the ER lumen by tunicamycin induced the ER stress response as evidenced by increased protein and gene expression of GADD153 as well as PERK and eIF2-alpha phosphorylation, and resulted in apoptosis. IL-1beta treatment induced PERK and eIF2-alpha phosphorylation, but not GADD153 expression or apoptosis. The ER stress signaling pathway of IL-1beta involved iNOS because blocking its expression, inhibited ER stress gene expression. Therefore, inducing the ER stress response in chondrocytes results in divergent responses depending on the agent used. Even though IL-1beta, a common proinflammatory cytokine, induces the ER stress response, it is not proapoptotic to chondrocytes. On the other hand, exposure to high levels of intracellular nitric oxide induce chondrocyte apoptosis as part of the ER stress response.     

Cellular basis of the role of diesel exhaust particles in inducing Th2-dominant response

There is growing evidence that diesel exhaust particles (DEP) can induce allergic diseases with increased IgE production and preferential activation of Th2 cells. To clarify the cellular basis of the role of DEP in the induction of Th2-dominant responses, we examined the effects of DEP on the cytokine production by T cells stimulated with anti-CD3/CD28 Ab and on that by monocyte-derived dendritic cells (MoDCs) stimulated with CD40L and/or IFN-gamma. We examined IFN-gamma, IL-4, IL-5, IL-8, and IL-10 produced by T cells and TNF-alpha, IL-1beta, IL-10, and IL-12 produced by MoDCs using real-time PCR analysis or by ELISA. To highlight the effects of DEP, we compared the effects of DEP with those of dexamethasone (DEX) and cyclosporin A (CyA). DEP significantly suppressed IFN-gamma mRNA expression and protein production, while it did not affect IL-4 or IL-5 mRNA expression or protein production. The suppressive effect on IFN-gamma mRNA expression was more potent than that of DEX and comparable at 30 mug/ml with 10(-7) M CyA. The suppressive effect on IFN-gamma production was also more potent than that of either DEX or CyA. DEP suppressed IL-12p40 and IL-12p35 mRNA expression and IL-12p40 and IL-12p70 production by MoDCs, while it augmented IL-1beta mRNA expression. Finally, by using a thiol antioxidant, N-acetyl cysteine, we found that the suppression of IFN-gamma production by DEP-treated T cells was mediated by oxidative stress. These data revealed a unique characteristic of DEP, namely that they induce a Th2 cytokine milieu in both T cells and dendritic cells.     

A new rhodopsin R135W mutation induces endoplasmic reticulum stress and apoptosis in retinal pigment epithelial cells

Rhodopsin mutations are associated with the autosomal-dominant form of retinitis pigmentosa (RP). Here we report simultaneous occurrence of RP associated with bilateral nanophthalmos and acute angle-closure glaucoma in patient with a new mutation in rhodopsin (R135W). ARPE-19 cells were transfected with myc-tagged wild-type (WT) and R135W rhodopsin constructs. The half-life of WT and R135W rhodopsin was analyzed via cycloheximide chase analysis. We found that R135W rhodopsin was accumulated in the endoplasmic reticulum (ER) and induced unfolded protein response (UPR) and apoptosis. Moreover, chaperone HSP70 alleviated ER stress and prevented apoptosis induced by R135W rhodopsin by attenuating UPR signaling. These findings reveal the novel pathogenic mechanism of RP and suggest that chaperone HSP70 has potential therapeutic significance for RP.     

The endoplasmic reticulum and unfolded protein response in the control of mammalian recombinant protein production

The endoplasmic reticulum (ER) of eukaryotic cells is involved in the synthesis and processing of proteins and lipids in the secretory pathway. These processing events that proteins undergo in the ER may present major limiting steps for recombinant protein production. Increased protein synthesis, accumulation of improperly processed or mis-folded protein can induce ER stress. To cope with ER stress, the ER has quality control mechanisms, such as the unfolded protein response (UPR) and ER-associated degradation to restore homeostasis. ER stress and UPR activation trigger multiple physiological cellular changes. Here we review cellular mechanisms that cope with ER stress and illustrate how this knowledge can be applied to increase the efficiency of recombinant protein expression.     

A novel cellular stress response characterised by a rapid reorganisation of membranes of the endoplasmic reticulum

Canonical endoplasmic reticulum (ER) stress, which occurs in many physiological and disease processes, results in activation of the unfolded protein response (UPR). We now describe a new, evolutionarily conserved cellular stress response characterised by a striking, but reversible, reorganisation of ER membranes that occurs independently of the UPR, resulting in impaired ER transport and function. This reorganisation is characterised by a dramatic redistribution and clustering of ER membrane proteins. ER membrane aggregation is regulated, in part, by anti-apoptotic BCL-2 family members, particularly MCL-1. Using connectivity mapping, we report the widespread occurrence of this stress response by identifying several structurally diverse chemicals from different pharmacological classes, including antihistamines, antimalarials and antipsychotics, which induce ER membrane reorganisation. Furthermore, we demonstrate the potential of ER membrane aggregation to result in pathological consequences, such as the long-QT syndrome, a cardiac arrhythmic abnormality, arising because of a novel trafficking defect of the human ether-a-go-go-related channel protein from the ER to the plasma membrane. Thus, ER membrane reorganisation is a feature of a new cellular stress pathway, clearly distinct from the UPR, with important consequences affecting the normal functioning of the ER.     

Proteasome inhibition induces differential heat shock protein response but not unfolded protein response in HepG2 cells

Liver, a central organ responsible for the metabolism of carbohydrates, proteins, and lipoproteins, is exposed to various kinds of physiological, pathological, and environmental stresses. We hypothesized that blockage of proteasome degradation pathway induces heat shock protein (HSP) response and unfolded protein response in the liver cells. In this study, we have characterized cellular responses to proteasome inhibition in HepG2 cells, a well-differentiated human hepatoma cells. We found that proteasome inhibition induced differential response among cytosolic HSPs, that is, increased expression of HSP70, but no change in HSP40, HSC70, and HSP90. However, proteasome inhibition did not induce typical unfolded protein response as indicated by absence of stimulation of GRP78 and GRP94 proteins. Upon proteasome inhibition, inclusion bodies were accumulated, and ubiquitin-conjugated proteins appeared in insoluble fraction, together with HSP40, HSP70, HSC70, and HSP90. After proteasome inhibition, misfolded proteins were increased in the cytosol and in the ER compartment as evaluated by examining ubiquitin-conjugated proteins. However, essentially all ER-associated ubiquitin-conjugated proteins were located on the surface of the ER, which explains why proteasome inhibition does not induce unfolded protein response. In conclusion, proteasome inhibition induces differential HSP response, but not unfolded protein response in HepG2 cells. Our study also suggests that HSPs play important roles in directing proteasomal degradation and protein aggregate formation.     

Palmitate and thapsigargin have contrasting effects on ER membrane lipid composition and ER proteostasis in neuronal cells

The endoplasmic reticulum (ER) is an organelle that performs several key functions such as protein synthesis and folding, lipid metabolism and calcium homeostasis. When these functions are disrupted, such as upon protein misfolding, ER stress occurs. ER stress can trigger adaptive responses to restore proper functioning such as activation of the unfolded protein response (UPR). In certain cells, the free fatty acid palmitate has been shown to induce the UPR. Here, we examined the effects of palmitate on UPR gene expression in a human neuronal cell line and compared it with thapsigargin, a known depletor of ER calcium and trigger of the UPR. We used a Gaussia luciferase-based reporter to assess how palmitate treatment affects ER proteostasis and calcium homeostasis in the cells. We also investigated how ER calcium depletion by thapsigargin affects lipid membrane composition by performing mass spectrometry on subcellular fractions and compared this to palmitate. Surprisingly, palmitate treatment did not activate UPR despite prominent changes to membrane phospholipids. Conversely, thapsigargin induced a strong UPR, but did not significantly change the membrane lipid composition in subcellular fractions. In summary, our data demonstrate that changes in membrane lipid composition and disturbances in ER calcium homeostasis have a minimal influence on each other in neuronal cells. These data provide new insight into the adaptive interplay of lipid homeostasis and proteostasis in the cell.     

Oxidative stress-induced expression of HSP70 contributes to the inhibitory effect of 15d-PGJ2 on inducible prostaglandin pathway in chondrocytes

The inhibitory effect of 15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂) on proinflammatory gene expression has been extensively documented and frequently ascribed to its ability to prevent NF-κB pathway activation. We and others have previously demonstrated that it was frequently independent of the peroxisome proliferator activated receptor (PPAR)γ activation. Here, we provide evidence that induction of intracellular heat shock protein (HSP)70 by oxidative stress is an additional regulatory loop supporting the anti-inflammatory effect of 15d-PGJ₂ in chondrocytes. Using real-time quantitative PCR and Western blotting, we showed that 15d-PGJ₂ stimulated HSP70, but not HSP27 expression while increasing oxidative stress as measured by spectrofluorimetry and confocal spectral imaging. Using N-acetylcysteine (NAC) as an antioxidant, we demonstrated further that oxidative stress was thoroughly responsible for the increased expression of HSP70. Finally, using an HSP70 antisense strategy, we showed that the inhibitory effect of 15d-PGJ₂ on IL-1-induced activation of the NF-κB pathway, COX-2 and mPGES-1 expression, and PGE₂ synthesis was partly supported by HSP70. These data provide a new anti-inflammatory mechanism to support the PPARγ-independent effect of 15d-PGJ₂ in chondrocyte and suggest a possible feedback regulatory loop between oxidative stress and inflammation via intracellular HSP70 up-regulation. This cross talk is consistent with 15d-PGJ₂ as a putative negative regulator of the inflammatory reaction.     

Endoplasmic reticulum stress and fungal pathogenesis

The gateway to the secretory pathway is the endoplasmic reticulum (ER), an organelle that is responsible for the accurate folding, post-translational modification and final assembly of up to a third of the cellular proteome. When secretion levels are high, errors in protein biogenesis can lead to the accumulation of abnormally folded proteins, which threaten ER homeostasis. The unfolded protein response (UPR) is an adaptive signaling pathway that counters a buildup in misfolded and unfolded proteins by increasing the expression of genes that support ER protein folding capacity. Fungi, like other eukaryotic cells that are specialized for secretion, rely upon the UPR to buffer ER stress caused by fluctuations in secretory demand. However, emerging evidence is also implicating the UPR as a central regulator of fungal pathogenesis. In this review, we discuss how diverse fungal pathogens have adapted ER stress response pathways to support the expression of virulence-related traits that are necessary in the host environment.     

Role of hyperhomocysteinemia in endothelial dysfunction and atherothrombotic disease

Hyperhomocysteinemia (HHcy) is an independent risk factor for cardiovascular disease, including ischemic heart disease, stroke, and peripheral vascular disease. Mutations in the enzymes responsible for homocysteine metabolism, particularly cystathionine beta-synthase (CBS) or 5,10-methylenetetrahydrofolate reductase (MTHFR), result in severe forms of HHcy. Additionally, nutritional deficiencies in B vitamin cofactors required for homocysteine metabolism, including folic acid, vitamin B6 (pyridoxal phosphate), and/or B12 (methylcobalamin), can induce HHcy. Studies using animal models of genetic- and diet-induced HHcy have recently demonstrated a causal relationship between HHcy, endothelial dysfunction, and accelerated atherosclerosis. Dietary enrichment in B vitamins attenuates these adverse effects of HHcy. Although oxidative stress and activation of proinflammatory factors have been proposed to explain the atherogenic effects of HHcy, recent in vitro and in vivo studies demonstrate that HHcy induces endoplasmic reticulum (ER) stress, leading to activation of the unfolded protein response (UPR). This review summarizes the current role of HHcy in endothelial dysfunction and explores the cellular mechanisms, including ER stress, that contribute to atherothrombosis.