Proteasome inhibition represses unfolded protein response and Nox4, sensitizing vascular cells to endoplasmic reticulum stress-induced death.

BACKGROUND: Endoplasmic reticulum (ER) stress has pathophysiological relevance in vascular diseases and merges with proteasome function. Proteasome inhibition induces cell stress and may have therapeutic implications. However, whether proteasome inhibition potentiates ER stress-induced apoptosis and the possible mechanisms involved in this process are unclear. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that proteasome inhibition with MG132, per se at non-lethal levels, sensitized vascular smooth muscle cells to caspase-3 activation and cell death during ER stress induced by tunicamycin (Tn). This effect was accompanied by suppression of both proadaptive (KDEL chaperones) and proapoptotic (CHOP/GADD153) unfolded protein response markers, although, intriguingly, the splicing of XBP1 was markedly enhanced and sustained. In parallel, proteasome inhibition completely prevented ER stress-induced increase in NADPH oxidase activity, as well as increases in Nox4 isoform and protein disulfide isomerase mRNA expression. Increased Akt phosphorylation due to proteasome inhibition partially offset the proapoptotic effect of Tn or MG132. Although proteasome inhibition enhanced oxidative stress, reactive oxygen species scavenging had no net effect on sensitization to Tn or MG132-induced cell death. CONCLUSION/RELEVANCE: These data indicate unfolded protein response-independent pathways whereby proteasome inhibition sensitizes vascular smooth muscle to ER stress-mediated cell death. This may be relevant to understand the therapeutic potential of such compounds in vascular disease associated with increased neointimal hyperplasia.

Protein disulfide isomerase (PDI) associates with NADPH oxidase and is required for phagocytosis of Leishmania chagasi promastigotes by macrophages.

PDI, a redox chaperone, is involved in host cell uptake of bacteria/viruses, phagosome formation, and vascular NADPH oxidase regulation. PDI involvement in phagocyte infection by parasites has been poorly explored. Here, we investigated the role of PDI in in vitro infection of J774 macrophages by amastigote and promastigote forms of the protozoan Leishmania chagasi and assessed whether PDI associates with the macrophage NADPH oxidase complex. Promastigote but not amastigote phagocytosis was inhibited significantly by macrophage incubation with thiol/PDI inhibitors DTNB, bacitracin, phenylarsine oxide, and neutralizing PDI antibody in a parasite redox-dependent way. Binding assays indicate that PDI preferentially mediates parasite internalization. Bref-A, an ER-Golgi-disrupting agent, prevented PDI concentration in an enriched macrophage membrane fraction and promoted a significant decrease in infection. Promastigote phagocytosis was increased further by macrophage overexpression of wild-type PDI and decreased upon transfection with an antisense PDI plasmid or PDI siRNA. At later stages of infection, PDI physically interacted with L. chagasi, as revealed by immunoprecipitation data. Promastigote uptake was inhibited consistently by macrophage preincubation with catalase. Additionally, loss- or gain-of-function experiments indicated that PMA-driven NADPH oxidase activation correlated directly with PDI expression levels. Close association between PDI and the p22phox NADPH oxidase subunit was shown by confocal colocalization and coimmunoprecipitation. These results provide evidence that PDI not only associates with phagocyte NADPH oxidase but also that PDI is crucial for efficient macrophage infection by L. chagasi.

Novel role of protein disulfide isomerase in the regulation of NADPH oxidase activity: pathophysiological implications in vascular diseases.

Vascular cell NADPH oxidase complexes are key sources of signaling reactive oxygen species (ROS) and contribute to disease pathophysiology. However, mechanisms that fine-tune oxidase-mediated ROS generation are incompletely understood. Besides known regulatory subunits, upstream mediators and scaffold platforms reportedly control and localize ROS generation. Some evidence suggest that thiol redox processes may coordinate oxidase regulation. We hypothesized that thiol oxidoreductases are involved in this process. We focused on protein disulfide isomerase (PDI), a ubiquitous dithiol disulfide oxidoreductase chaperone from the endoplasmic reticulum, given PDI's unique versatile role as oxidase/isomerase. PDI is also involved in protein traffic and can translocate to the cell surface, where it participates in cell adhesion and nitric oxide internalization. We recently provided evidence that PDI exerts functionally relevant regulation of NADPH oxidase activity in vascular smooth muscle and endothelial cells, in a thiol redox-dependent manner. Loss-of-function experiments indicate that PDI supports angiotensin II-mediated ROS generation and Akt phosphorylation. In addition, PDI displays confocal co-localization and co-immunoprecipitates with oxidase subunits, indicating close association. The mechanisms of such interaction are yet obscure, but may involve subunit assembling stabilization, assistance with traffic, and subunit disposal. These data may clarify an integrative view of oxidase activation in disease conditions, including stress responses.

Persistent activation of Akt or ERK prevents the toxicity induced by saturated and polyunsaturated fatty acids in RINm5F ß-cells.

The aim of this study was to investigate whether the toxicity of saturated and polyunsaturated fatty acids (PUFA) on RINm5F cells is related to the phosphorylation state of Akt, ERK and PKC delta. The regulation of these kinases was compared in three experimental designs: (a) 4h-exposure, (b) 4h-exposure and a subsequent withdrawn of the FA for a 20 h period and (c) 24h-exposure. Saturated and PUFA were toxic to RINm5F cells even at low concentrations. Also, evidence is provided for a late (i.e. the effect only appeared hours after the treatment) and a persistent regulation (i.e. maintenance of the effect for several hours) of Akt, ERK and PKC delta phosphorylation by the FA. Late activation of PKC delta seems important for palmitate cytotoxicity. Persistent activation of the survival proteins Akt and ERK by stearate, oleate and arachidonate might play an important role to prevent the toxic effect of posterior PKC delta activation. The results shown may explain why a short-period exposure to FA is not enough to induce cytotoxicity in pancreatic beta-cells, since survival pathways are activated. Besides, when this activation is persistent, it may overcome a posterior induction of death pathways.

Distinct regulation of IRS proteins in adipose tissue from obese aged and dexamethasone-treated rats.

In the present study, we investigated the protein levels and phosphorylation status of the insulin receptor and insulin receptor substrates (IRS-1, IRS-2, and IRS-3) as well as their association with PI(3)-kinase in the rat adipose tissue of two models of insulin resistance: dexamethasone treatment and aging. AKT and atypical PKC phosphorylation detection were also performed. Both models showed decreased insulin-induced IRS-1 and IRS-2 tyrosine phosphorylation, accompanied by reduced protein levels of IRS-1 and IRS-2. Nevertheless, IRS-3 protein level was unchanged in aging but increased in dexamethasone-treated rats. PI(3)-kinase association with IRS-1 was reduced in aged rats, whereas dexamethasone-treated rats showed a reduced IRS-2/ PI(3)-kinase association. However, IRS-3 association with PI(3)-kinase was reduced in both models, as well as insulin-induced AKT and PKC phosphorylation. The alterations described in the present study show that the action of insulin is differently impaired depending on the origin of insulin resistance. These differences might be directly linked to the singular metabolic features of the models we tested.

Dehydroepiandrosterone increases beta-cell mass and improves the glucose-induced insulin secretion by pancreatic islets from aged rats.

The effect of dehydroepiandrosterone (DHEA) on pancreatic islet function of aged rats, an animal model with impaired glucose-induced insulin secretion, was investigated. The following parameters were examined: morphological analysis of endocrine pancreata by immunohistochemistry; protein levels of insulin receptor, IRS-1, IRS-2, PI 3-kinase, Akt-1, and Akt-2; and static insulin secretion in isolated pancreatic islets. Pancreatic islets from DHEA-treated rats showed an increased beta-cell mass accompanied by increased Akt-1 protein level but reduced IR, IRS-1, and IRS-2 protein levels and enhanced glucose-stimulated insulin secretion. The present results suggest that DHEA may be a promising drug to prevent diabetes during aging.

Nitrogen dioxide and carbonate radical anion: two emerging radicals in biology.

Nitrogen dioxide and carbonate radical anion have received sporadic attention thus far from biological investigators. However, accumulating data on the biochemical reactions of nitric oxide and its derived oxidants suggest that these radicals may play a role in various pathophysiological processes. These potential roles are also indicated by recent studies on the high efficiency of urate and nitroxides in protecting cells and whole animals against the injury associated with conditions of excessive nitric oxide production. The high protective effects of these antioxidants are incompletely defined at the mechanistic level but some of them can be explained by their efficiency in scavenging peroxynitrite-derived radicals, particularly nitrogen dioxide and carbonate radical anion. In this review, we provide a framework for this hypothesis and discuss the potential sources and properties of these radicals that are likely to become increasingly recognized as important mediators of biological processes.