Alexandre F. Fernandes

Sustained oxidative stress inhibits NF-κB activation partially via inactivating the proteasome

NF-kappaB is a family of important transcription factors involved in many cellular functions, such as cell survival, proliferation, and stress responses. Many studies indicate that NF-kappaB is a stress-sensitive transcription factor and its activation is regulated by reactive oxygen species. In previous studies, we and others demonstrated that this transcription factor can be activated by transient oxidative stress. However, the effects of sustained oxidative stress on NF-kappaB activation are not clear. The objective of this study was to determine the effects of sustained oxidative stress on NF-kappaB activation and to elucidate the signaling events affected by sustained oxidative stress. Human lens epithelial cells (HLEC) that were subjected to 4 h of continuous influx of hydrogen peroxide were used to investigate the effects of sustained oxidative stress on NF-kappaB activation. The data showed that, unlike transient oxidative stress, sustained exposure of HLEC to physiologically relevant levels of H(2)O(2) (50-100 microM for 4 h) did not induce the degradation of I-kappaB and activation of NF-kappaB, but attenuated TNFalpha-induced degradation of I-kappaB and activation of NF-kappaB. Sustained exposure of HLEC to these levels of H(2)O(2) also inactivated proteasome activity by 50-80%. Consistent with the role of the proteasome in degradation of I-kappaB and activation of NF-kappaB, treatment of HLEC with proteasome inhibitors also attenuated TNFalpha-induced I-kappaB degradation and NF-kappaB activation. The data also indicate that activation of NF-kappaB is essential for the cells to recover from oxidative stress. Inhibiting NF-kappaB activation during recovery from transient oxidative stress significantly reduced the cell viability. Together, these data indicate that sustained oxidative stress may inactivate the proteasome and subsequently inhibit NF-kappaB activation by impeding the degradation of I-kappaB. The oxidative inactivation of the proteasome and subsequent impairment of NF-kappaB activation may contribute to the death of lens epithelial cells, a common feature associated with cataract.

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

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 H2O2, 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-κB and p38 MAPK. Proteasome inhibition both attenuated the activation and delayed the turnoff of NF-κB, 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.

The proteasome: a target of oxidative damage in cultured human retina pigment epithelial cells.

PURPOSE Dysfunction of the ubiquitin-proteasome pathway (UPP) is associated with several age-related degenerative diseases. The objective of this study was to investigate the effect of oxidative stress on the UPP in cultured human retina pigment epithelial cells. METHODS To mimic physiological oxidative stress, ARPE-19 cells were exposed to continuously generated H2O(2) or A2E-mediated photooxidation. Proteasome activity was monitored using fluorogenic peptides as substrates. The ubiquitin conjugation activity and activities of E1 and E2 were determined by the thiolester assays. Levels of ubiquitin and ubiquitin conjugates were determined by Western blotting. RESULTS Exposure of ARPE-19 cells to 40 to 50 microM H2O(2) for 4 hours resulted in a 30% to 50% reduction in all three peptidase activities of the proteasome. Similarly, exposure of A2E-loaded ARPE-19 cells to blue light resulted in a 40% to 60% reduction in proteasome activity. Loading of A2E or exposure to blue light alone had little effect on proteasome activity. In contrast, exposure of ARPE-19 to low levels of H2O(2) (10 microM) stimulated ubiquitin conjugation activity. Loading of A2E, with or without exposure to blue light, upregulated the levels of ubiquitin-activating enzyme and increased conjugation activity. Exposure to H2O(2) or A2E-mediated photooxidation also resulted in a twofold to threefold increase in levels of endogenous ubiquitin conjugates. CONCLUSIONS These data show that the proteasome in ARPE-19 is susceptible to oxidative inactivation, whereas activities of the ubiquitin-conjugating enzymes are more resistant to oxidative stress. Oxidative inactivation of the proteasome appears to be one of the mechanisms underlying stress-induced accumulation of ubiquitin conjugates in the cells.

Proteasome Inactivation Promotes p38 Mitogen-activated Protein Kinase-dependent Phosphatidylinositol 3-kinase Activation and Increases Interleukin-8 Production in Retinal Pigment Epithelial Cells

Oxidative stress and inflammation are implicated in the pathogenesis of many age-related diseases. We have demonstrated previously that oxidative inactivation of the proteasome is a molecular link between oxidative stress and overexpression of interleukin (IL)-8. Here, we elucidated a novel signaling cascade that leads to up-regulation of IL-8 in response to proteasome inactivation. The sequence of events in this cascade includes proteasome inactivation, activation of mitogen-activated protein kinase kinase (MKK)3/MKK6, activation of p38 mitogen-activated protein kinase (MAPK), epidermal growth factor receptor phosphorylation, phosphatidylinositol 3-kinase (PI3K) activation and increased IL-8 expression. Blocking any of these signaling pathways abolished the up-regulation of IL-8 induced by proteasome inhibition. Although Akt is also activated in response to proteasome inactivation, we found that the PI3K-dependent up-regulation of IL-8 is independent of 3-phosphoinositide-dependent protein kinase (PDK)1 and Akt. Inhibition of PDK1 and Akt with chemical inhibitors or expression of constitutive active Akt had little effects on IL-8 expression in response to proteasome inactivation. In contrast, inhibition of interleukin 2-inducible T cell kinase, a kinase downstream of PI3K, significantly reduced the expression and secretion of IL-8 in response to proteasome inactivation. Together, these data elucidate a novel signaling network that leads to increased IL-8 production in response to proteasome inactivation.