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.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[04/13683-0]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[09/54764-6]Instituto Nacional de Ciencia e Tecnologia de Processos Redox em BioMedicina (Redoxoma, INCT, CNPq)Fundacao Zerbin

Effect of the Antioxidant Lipoic Acid in Aortic Phenotype in a Marfan Syndrome Mouse Model

Marfan syndrome (MFS) cardiovascular manifestations such as aortic aneurysms and cardiomyopathy carry substantial morbidity/mortality. We investigated the effects of lipoic acid, an antioxidant, on ROS production and aortic remodeling in a MFS mgΔloxPneo mouse model. MFS and WT (wild-type) 1-month-old mice were allocated to 3 groups: untreated, treated with losartan, and treated with lipoic acid. At 6 months old, echocardiography, ROS production, and morphological analysis of aortas were performed. Aortic ROS generation in 6-month-old MFS animals was higher at advanced stages of disease in MFS. An unprecedented finding in MFS mice analyzed by OCT was the occurrence of focal inhomogeneous regions in the aortic arch, either collagen-rich extremely thickened or collagen-poor hypotrophic regions. MFS animals treated with lipoic acid showed markedly reduced ROS production and lower ERK1/2 phosphorylation; meanwhile, aortic dilation and elastic fiber breakdown were unaltered. Of note, lipoic acid treatment associated with the absence of focal inhomogeneous regions in MFS animals. Losartan reduced aortic dilation and elastic fiber breakdown despite no change in ROS generation. In conclusion, oxidant generation by itself seems neutral with respect to aneurysm progression in MFS; however, lipoic acid-mediated reduction of inhomogeneous regions may potentially associate with less anisotropy and reduced chance of dissection/rupture

Signaling patways redox involved in the differentiation of human monocytes to macrophages

O processo de diferenciacao de celulas hematopoieticas consiste de varios eventos. A participacao de citocinas e essencial para que cada celula, proveniente de uma mesma matriz, se diferencie ate seu estagio final. Os monocitos sao capazes de se diferenciarem a macrofagos atraves de estimulos que promovem modificacoes moleculares, definindo as alteracoes funcionais e morfologicas. Estudos previos demonstraram que alteracoes no status redox intracelular com predominancia de uma condicao oxidante, sao fundamentais para que a diferenciacao ocorra. Sabe-se tambem que algumas das proteinas quinases que participam do l processo,JNK,ERKe Pyk2,sao sensiveis a estas alteracoes dostatusredoxl intracelular. Celulas da linhagem monocitica humana (THP1) foram utilizadas como modelo para a caracterizacao das vias de sinalizacao envolvidas) na diferenciacao de monocitos a macrofagos.Como indutor da diferenciacao foi utilizado o PMA, um ester de forbol, capaz de ativar a PKC e gerar estresse oxidativo endogeno. Para analisarmos a influencia da alteracao do ambiente redox intracelular, submetemos as celulas a um pre-tratamento com moduladores positivos e negativos da sintese de glutationa (NAC e BSO, respectivamente) seguido do estimulo com PMAa(au)BV UNIFESP: Teses e dissertaçõe

Cellular prion protein (PrP(C)) and superoxide dismutase (SOD) in vascular cells under oxidative stress

The PrP(C) is expressed in several cell types but its physiological function is unknown. Some studies associate the PrP(C) with copper metabolism and the antioxidant activity of SOD. Our hypothesis was that changes in PrP(C) expression lead to abnormal copper regulation and induce SOD downregulation in the vascular wall. Objectives: to study whether the PrP(C) expression undergoes induction by agents that trigger endoplasmic reticulum stress (ERS) and, in this context, to evaluate the SOD activity. Methods: To trigger ERS, in vitro, rabbit aortic smooth muscle cells were challenged for 4, 8 and 18 hours, with angiotensin-II, tunicamycin and 7-ketocholesterol. For in vivo studies rabbit aortic arteries were subjected to injury by balloon catheter. Results: In vitro baseline SOD activity, determined through inhibition of cytochrome-c reduction, was 13.9 +/- 1.2 U/mg protein, angiotensin-II exposed for 8 hours produced an increase in SOD activity, and cellular copper concentration was about 9 times greater only under these conditions. Western blotting analysis for SOD isoenzymes showed an expression profile that was not correlated with the enzymatic activity. PrP(C) expression decreased after exposure to all agents after different incubation periods. RT-PCR assay showed increased mRNA expression for PrP(C) only in cells stimulated for 8 hours with the different stressors. The PrP(C) mRNA expression in rabbit aortic artery fragments, subjected to balloon catheter injury, showed a pronounced increase immediately after overdistension. The results obtained indicated a PrP(C) protection factor during the early part of the ERS exposure period, but did not demonstrate a SOD-like profile for the PrP(C). (C) 2009 Elsevier GmbH. All rights reserved.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) of Brazi

Proteasome inhibition sensitizes VSMC to death due to ER stress.

<p>(A) Representative graph of VSMC cell viability by MTT assay. VSMC were incubated with tunicamycin (Tn) (5 µg/mL) or/and MG132 (1 µM) for 16 h, followed by MTT assays. (B) Similar to A, VSMC were incubated with Tn (5 µg/mL) or/and MG132 (1 µM) for 16 hours. Total cell homogenates were submitted to western analysis with anti-caspase-3 antibody. Data are mean ± SD of 3 independent experiments.*P<0.05 vs. Control. #P<0.05 vs.Tn.</p

Regulation of p21Waf1 expression and TNF alpha(biosynthesis by glutathione modulators in PMA induced-THP1 differentiation: Involvement of JNK and ERK pathways

Oxidative modifications of proteins are fundamental biochemical events that regulate cellular signaling, protein expression, and function. the redox status is balanced by reductants in which GSH plays a major role. This study investigated whether or not p21Waf1 expression and TNF alpha biosynthesis in macrophage differentiation/activation were regulated by GSH modulators and whether or not the JNK and ERK pathway were involved. We observed an increase of p21Waf1 expression and TNF alpha biosynthesis in the THP1 monocyte/macrophage cell line treated with PMA. Treatment of THP1 cultures with NAC prior to adding PMA abrogates the expression of p21Waf1 mRNA and decreases the level of TNF alpha whereas GSH depletion by BSO enhances the levels of TNF alpha with minor effects on p21Waf1 expression. To assess whether or not ERK and JNK were involved in the redox mechanism of p21Waf1 and TNF alpha, we used pharmacological inhibitors for JNK and ERK. Both PD98095 and dicoumarol were capable of blocking TNF alpha production but had only a small effect on p21Waf1 expression. We next observed that activation of JNK was significantly inhibited in cells pretreated with NAC with no effect on ERK. Taken together, our findings suggest that the modulation of GSH regulate the magnitude the cell response to PMA in which JNK and ERK have a particular role in redox signaling, (C) 2007 Elsevier Inc. All rights reserved.Universidade Federal de São Paulo, Escola Paulista Med, CINTERGEN, Dept Bioquim & Biol Mol, São Paulo, BrazilUniv São Paulo, Inst Coracao, São Paulo, BrazilUniv São Paulo, Fac Ciencias Farmaceut, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Clin Med, São Paulo, BrazilNYU, Sch Med, Dept Pharmacol, New York, NY USAUniversidade Federal de São Paulo, Escola Paulista Med, CINTERGEN, Dept Bioquim & Biol Mol, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Clin Med, São Paulo, BrazilWeb of Scienc

Activity of 20S proteasome in VSMC at control condition or after incubation with AngII (200 nM) or Tn (5 µg/mL), in the absence or presence of MG132 (1 µM) during 4 (A) or 16 (B) hours.

<p>Cell lysates were incubated with probe AMC (LLVY-AMC) in the presence of SDS and fluorescence release followed over 15–30 min (excitation 355 nm, emission 460 nm). Data are mean ± SD of 4 or more independent experiments.*P<0.05 vs. Control. #P<0.05 vs. Tn.</p

Down-regulation of UPR signaling by MG132.

<p>VSMC were incubated with Tn (5 µg/ml), MG132 (1 µM) or their combination for 16 h. Total cell homogenates were submitted to western analysis with anti-KDEL (A) or anti-PDI antibodies (B). Graphs to the right are corresponding densitometric measurements of blots shown in (A) and (B) for at least 3 independent experiments; (C) Analysis of PDI mRNA by real-time PCR. VSMC were incubated with vehicle or Tn (5 µg/mL) in the absence or presence of MG132 (1 µM) for 16 h; n = 3. Data are mean ± SD. *P<0.05 vs. Control. #P<0.05 vs.Tn.</p

Proteasome inhibition induces ROS production and disrupts ER stress-induced NADPH oxidase up-regulation.

<p>(A) ROS production in VSMC incubated for 4 h with Tn (5 µg/mL) or/and MG132 (1 µM), assessed through HPLC analysis of DHE oxidation products (50 µM, 30 min incubation), as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014591#s4" target="_blank">Methods</a>. Results depict levels of 2-hydroxyethidium (EOH) or ethidium (E) products. (B) NADPH oxidase activity measured in membrane-enriched homogenates from VSMC incubated for 4 h with Tn or MG132. Activity was measured with DHE technique, analogous to (A), as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014591#s4" target="_blank">Methods</a>. Data are mean ± SD of 3 independent experiments.*P<0.05 vs. Control. #P<0.05 vs.Tn.</p

Effects of ROS scavenging in VSMC viability after ER stress or/and proteasome inhibition.

<p>VSMC were incubated with Tn (5 µg/mL) or/and MG132 (1 µM) for 16 h in the absence or presence of PEG-Cat (200 U/ml) plus PEG-SOD (25 U/ml). Data are mean ± SD of 3 independent experiments.*P<0.05 vs. Control. #P<0.05 vs.Tn.</p