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Oxidative inactivation of the proteasome in retinal pigment epithelial cells. A potential link between oxidative stress and up-regulation of interleukin-8.

ABSTRACT: Oxidative stress and inflammation are implicated in the pathogenesis of many age-related diseases. Stress-induced overproduction of inflammatory cytokines, such as interleukin-8 (IL-8), is one of the early events of inflammation. The objective of this study was to elucidate mechanistic links between oxidative stress and overproduction of IL-8 in retinal pigment epithelial (RPE) cells. We found that exposure of RPE cells to H(2)O(2), paraquat, or A2E-mediated photooxidation resulted in increased expression and secretion of IL-8. All of these oxidative stressors also inactivated the proteasome in RPE cells. In contrast, tert-butylhydroperoxide (TBH), a lipophilic oxidant that did not stimulate IL-8 production, also did not inactivate the proteasome. Moreover, prolonged treatment of RPE cells with proteasome-specific inhibitors recapitulated the stimulation of IL-8 production. These data suggest that oxidative inactivation of the proteasome is a potential mechanistic link between oxidative stress and up-regulation of the proinflammatory IL-8. The downstream signaling pathways that govern the production of IL-8 include NF-kappaB and p38 MAPK. Proteasome inhibition both attenuated the activation and delayed the turnoff of NF-kappaB, resulting in biphasic effects on the production of IL-8. Prolonged proteasome inhibition (>2 h) resulted in activation of p38 MAPK via activation of MKK3/6 and increased the production of IL-8. Chemically inhibiting the p38 MAPK blocked the proteasome inhibition-induced up-regulation of IL-8. Together, these data indicate that oxidative inactivation of the proteasome and the related activation of the p38 MAPK pathway provide a potential link between oxidative stress and overproduction of proinflammatory cytokines, such as IL-8.

SUBMITTER: Fernandes AF

PROVIDER: S-EPMC2475710 | biostudies-literature | 2008 Jul

REPOSITORIES: biostudies-literature

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Oxidative inactivation of the proteasome in retinal pigment epithelial cells. A potential link between oxidative stress and up-regulation of interleukin-8.

Fernandes Alexandre F AF Zhou Jilin J Zhang Xinyu X Bian Qingning Q Sparrow Janet J Taylor Allen A Pereira Paulo P Shang Fu F

The Journal of biological chemistry 20080523 30

Oxidative stress and inflammation are implicated in the pathogenesis of many age-related diseases. Stress-induced overproduction of inflammatory cytokines, such as interleukin-8 (IL-8), is one of the early events of inflammation. The objective of this study was to elucidate mechanistic links between oxidative stress and overproduction of IL-8 in retinal pigment epithelial (RPE) cells. We found that exposure of RPE cells to H(2)O(2), paraquat, or A2E-mediated photooxidation resulted in increased ...[more]

PMID: 18502748

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Project description:PurposeIn this work, we investigated the ability of pinosylvin (PS), 3,5-dihydroxy-trans-stilbene, to modulate oxidative stress in human RPE cells. PS, a stilbenoid polyphenol, occurs in high concentrations in bark byproducts and therefore represents an attractive bioactive compound for health-promoting applications.MethodsFirst, we evaluated the toxicity range of PS by exposing ARPE-19 cells to 0.1-200 µM concentrations of PS for 24 h followed by the cell viability test. In the next stage, the ARPE-19 cells were preincubated in PS for 24 h followed by hydroquinone (HQ) exposure without PS for another 24 h. The cell viability test was conducted after HQ exposure. To elucidate the potential mechanisms behind PS-mediated protection against oxidative stress, the ARPE-19 cells were treated with 5 µM PS for 6 h, and mRNA was extracted at four time points (2 h, 6 h, 12 h, 24 h) to determine changes in the expression of nuclear factor-erythroid 2-related factor-2 (Nrf2), sequestosome 1 (p62/SQSTM1), heme oxygenase-1 (HO-1), and glutathione S-transferase pi 1 (GSTP1) genes. To clarify the molecular mechanism behind PS-mediated protection further, the ARPE-19 cells were transfected with p62 and Nrf2 siRNAs for 24 h, and the roles of p62, Nrf2, and its target gene HO-1 in conferring protection against oxidative stress were studied with quantitative real-time PCR (qRT-PCR) and the cell viability test.ResultsPS treatment at concentrations of 5 and 10 µM significantly enhanced cell survival from oxidative stress. The expression levels of an enzyme with antioxidative, anti-inflammatory, and immunomodulatory properties, HO-1, were increased by PS treatment and correlated strongly with cell survival. PS treatment did not elevate the expression levels of Nrf2 or its target genes, p62 or GSTP1, even though it had a clear effect on the expression of HO-1, another gene controlled by Nrf2. RNA interference analysis further confirmed the important role of Nrf2 and HO-1 in PS-mediated protection against oxidative stress whereas the role of p62 seemed to be insignificant at the gene expression and cell viability levels.ConclusionsOur results suggest that PS treatment conferred protection against oxidative stress through the induction of HO-1 in human RPE cells. Consequently, PS-stilbene compounds, which can be isolated in significant amounts from bark waste, may possess health-promoting properties against aging-related diseases associated with oxidative stress such as age-related macular degeneration (AMD) and Alzheimer's disease. These natural compounds may offer opportunities for high-value use of bark waste in diverse health-related applications.

Project description:PurposeTo study the effect of subtoxic levels of hydrogen peroxide (H(2)O(2)) on the expression and release of interleukin-6 (IL-6) by cultured retinal pigment epithelial (RPE) cells and to explore the relevant signal pathways.MethodsCultured human RPE cells were stimulated with various subtoxic concentrations of H(2)O(2) for different periods. Conditioned medium and cells were collected. IL-6 in the medium and IL-6 mRNA in the collected cells were measured using an IL-6 enzyme-linked immunosorbent assay kit and reverse transcriptase polymerase chain reaction, respectively. Nuclear factor-kappaB (NF-κB) in nuclear extracts and phosphorylated p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinases (JNK) in cells cultured with and without H(2)O(2) were measured by NF-κB and MAPK enzyme-linked immunosorbent assay kits. Inhibitors of p38 (SB203580), ERK (UO1026), JNK (SP600125), and NF-κB (BAY11-7082) were added to the cultures before the addition of H(2)O(2) to test their effects(.)ResultsSubtoxic levels of H(2)O(2) (100 µM and less) increased the IL-6 mRNA level and the release of IL-6 protein by the cultured human RPE cells in a dose- and time-dependent manner. This was accompanied by an increase of NF-κB in nuclear extracts and phosphorylated p38 MAPK, ERK, and JNK in cell lysates, particularly in the p38 and NF-κB. The NF-κB inhibitor decreased the H(2)O(2)-induced expression of IL-6. The p38 inhibitor, but not the ERK or JNK inhibitor, completely abolished H(2)O(2)-induced expression of IL-6 by RPE cells. The p38 inhibitor also abolished the increase of NF-κB in nuclear extracts in cells treated with H(2)O(2).ConclusionsH(2)O(2) stimulated the production of IL-6, a key factor in the modulation of immune responses, inflammatory processes, and the occurrence of autoimmune diseases, which recently has been documented to be increased in age-related macular degeneration (AMD). This may be a molecular linkage for the oxidative stress and inflammatory/autoimmune reactions in AMD and may provide a novel target for the treatment of AMD.

Project description:Age-related macular degeneration (AMD) is a degenerative disease of the retina and the leading cause of blindness in the elderly. Retinal pigment epithelial (RPE) cell death and the resultant photoreceptor apoptosis are characteristic of late-stage dry AMD, especially geographic atrophy (GA). Although oxidative stress and inflammation have been associated with GA, the nature and underlying mechanism for RPE cell death remains controversial, which hinders the development of targeted therapy for dry AMD. The purpose of this study is to systematically dissect the mechanism of RPE cell death induced by oxidative stress. Our results show that characteristic features of apoptosis, including DNA fragmentation, caspase 3 activation, chromatin condensation and apoptotic body formation, were not observed during RPE cell death induced by either hydrogen peroxide or tert-Butyl hydroperoxide. Instead, this kind of cell death can be prevented by RIP kinase inhibitors necrostatins but not caspase inhibitor z-VAD, suggesting necrotic feature of RPE cell death. Moreover, ATP depletion, receptor interacting protein kinase 3 (RIPK3) aggregation, nuclear and plasma membrane leakage and breakdown, which are the cardinal features of necrosis, were observed in RPE cells upon oxidative stress. Silencing of RIPK3, a key protein in necrosis, largely prevented oxidative stress-induced RPE death. The necrotic nature of RPE death is consistent with the release of nuclear protein high mobility group protein B1 into the cytoplasm and cell medium, which induces the expression of inflammatory gene TNF? in healthy RPE and THP-1 cells. Interestingly, features of pyroptosis or autophagy were not observed in oxidative stress-treated RPE cells. Our results unequivocally show that necrosis, but not apoptosis, is a major type of cell death in RPE cells in response to oxidative stress. This suggests that preventing oxidative stress-induced necrotic RPE death may be a viable approach for late-stage dry AMD.

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Oxidative inactivation of the proteasome in retinal pigment epithelial cells. A potential link between oxidative stress and up-regulation of interleukin-8.

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Oxidative inactivation of the proteasome in retinal pigment epithelial cells. A potential link between oxidative stress and up-regulation of interleukin-8.

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