Qingning Bian

Lutein and zeaxanthin supplementation reduces photooxidative damage and modulates the expression of inflammation-related genes in retinal pigment epithelial cells

Oxidative damage and inflammation are related to the pathogenesis of age-related macular degeneration (AMD). Epidemiologic studies suggest that insufficient dietary lutein and zeaxanthin intake or lower serum zeaxanthin levels are associated with increased risk for AMD. The objective of this work is to test the protective effects of lutein and zeaxanthin against photooxidative damage to retinal pigment epithelial cells (RPE) and oxidation-induced changes in expression of inflammation-related genes. To mimic lipofuscin-mediated photooxidation in vivo, we used ARPE-19 cells that accumulated A2E, a lipofuscin fluorophore and photosensitizer, as a model system to investigate the effects of lutein and zeaxanthin supplementation. The data show that supplementation with lutein or zeaxanthin in the medium resulted in accumulation of lutein or zeaxanthin in the RPE cells. The concentrations of lutein and zeaxanthin in the cells were 2- to 14-fold of that detected in the medium, indicating that ARPE-19 cells actively take up lutein or zeaxanthin. As compared with untreated cells, exposure of A2E-containing RPE to blue light resulted in a 40-60% decrease in proteasome activity, a 50-80% decrease in expression of CFH and MCP-1, and an~20-fold increase in expression of IL-8. The photooxidation-induced changes in expression of MCP-1, IL-8, and CFH were similar to those caused by chemical inhibition of the proteasome, suggesting that inactivation of the proteasome is involved in the photooxidation-induced alteration in expression of these inflammation-related genes. Incubation of the A2E-containing RPE with lutein or zeaxanthin prior to blue light exposure significantly attenuated the photooxidation-induced inactivation of the proteasome and photooxidation-induced changes in expression of MCP-1, IL-8, and CFH. Together, these data indicate that lutein or zeaxanthin modulates inflammatory responses in cultured RPE in response to photooxidation. Protecting the proteasome from oxidative inactivation appears to be one of the mechanisms by which lutein and zeaxanthin modulate the inflammatory response. Similar mechanisms may explain salutary effects of lutein and zeaxanthin in reducing the risk for AMD.

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.

Natural History of Age-Related Retinal Lesions That Precede AMD in Mice Fed High or Low Glycemic Index Diets

PURPOSE Epidemiologic data indicate that people who consume low glycemic index (GI) diets are at reduced risk for the onset and progression of age-related macular degeneration (AMD). The authors sought corroboration of this observation in an animal model. METHODS Five- and 16-month-old C57BL/6 mice were fed high or low GI diets until they were 17 and 23.5 months of age, respectively. Retinal lesions were evaluated by transmission electron microscopy, and advanced glycation end products (AGEs) were evaluated by immunohistochemistry. RESULTS Retinal lesions including basal laminar deposits, loss of basal infoldings, and vacuoles in the retinal pigment epithelium were more prevalent in the 23.5- than in the 17-month-old mice. Within each age group, consumption of a high GI diet increased the risk for lesions and the risk for photoreceptor abnormalities and accumulation of AGEs. CONCLUSIONS Consuming high GI diets accelerates the appearance of age-related retinal lesions that precede AMD in mice, perhaps by increasing the deposition of toxic AGEs in the retina. The data support the hypothesis that consuming lower GI diets, or simulation of their effects with nutraceuticals or drugs, may protect against AMD. The high GI-fed C57BL/6 mouse is a new model of age-related retinal lesions that precede AMD and mimic the early stages of disease and may be useful for drug discovery.

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.

Lutein or Zeaxanthin Supplementation Suppresses Inflammatory Responses in Retinal Pigment Epithelial Cells and Macrophages

Recent studies indicate that an excessive inflammatory response is etiologically related to the pathogenesis of age-related macular degeneration (AMD). Epidemiologic studies suggest that dietary lutein and zeaxanthin intake is inversely associated systemic inflammation and risk for AMD. The objective of this work is to directly determine the effects of lutein and zeaxanthin supplementation on ocular and systemic inflammatory response. ARPE-19 cells and primary murine macrophages were cultured in the presence of 0, 1 and 10 μM lutein or zeaxanthin for 1–3 days and then stimulated with lipopolysaccharide (LPS) in fresh medium for 8 h. Levels of IL-6, IL-8 and TNFα in the medium were determined by ELISA and used as indicators of inflammatory response. To test the anti-inflammatory effects of lutein/zeaxanthin in vivo, C57BL mice were fed diets containing 0, 0.02%, and 0.1% lutein or zeaxanthin for 3 months. Macrophages were isolated from peritonea and cultured in the presence of LPS for 16 h. Levels of IL-6 and TNFα in the medium were determined. Cultured RPE produced relatively high levels of IL-6 and IL-8 even in the absence of LPS, but levels of IL-6 and TNF〈 secreted by macrophages in resting state were barely detectable. Treatment of RPE and macrophages with LPS resulted in a dramatic increase in production of IL-6, IL-8, and TNFα. Supplementation with lutein suppressed LPS-stimulated production of these inflammatory mediators in both RPE and macrophages. These suppressive effects were dose dependent. Lutein supplementation to cultured RPE also suppressed the production of IL-6 and IL-8 in the absence of LPS stimulation. Furthermore, macrophages isolated from lutein-supplemented mice produced less IL-6 and TNFα upon LPS stimulation and the suppressive effects were also dose dependent. Supplementation with zeaxanthin suppressed the production of IL-8, but not IL-6. These data indicate that lutein and zeaxanthin have anti-inflammatory effects and that increased dietary lutein or zeaxanthin intake may reduce the risk for AMD via modulating ocular and systemic inflammation.

Oligomerization with wt αA- and αB-crystallins reduces proteasome-mediated degradation of C-terminally truncated αA-crystallin.

PURPOSE We previously demonstrated that the ubiquitin-proteasome pathway (UPP) is a general protein quality control system that selectively degrades damaged or abnormal lens proteins, including C-terminally truncated αA-crystallin. The objective of this work was to determine the effects of wt αA- and αB-crystallins on the degradation of C-terminally truncated αA-crystallin (αA(1-162)) and vice versa. METHODS Recombinant wt αA, αB, and αA(1-162) were expressed in Escherichia coli and purified to homogeneity by chromatography. Subunit exchange and oligomerization were detected by fluorescence resonance energy transfer (FRET), multiangle-light scattering and coprecipitation assays. Protein substrates were labeled with (125)I and lens epithelial cell lysates were used as the source of the UPP for degradation assays. RESULTS FRET, multiangle light scattering, and coprecipitation assays showed that αA(1-162) exchanged subunits with wt αA- or wt αB- crystallin to form hetero-oligomers. αA(1-162) was more susceptible than wt αA-crystallin to degradation by the UPP. When mixed with wt αA-crystallin at 1:1 or 1:4 (αA(1-162) : wt) ratios to form hetero-oligomers, the degradation of αA(1-162) was significantly decreased. Conversely, formation of hetero-oligomers with αA(1-162) enhanced the degradation of wt αA-crystallin. The presence of αA(1-162), but not wt αA-crystallin, decreased the degradation of wt αB-crystallin. CONCLUSIONS αA(1-162) forms hetero-oligomers with wt αA- and αB-crystallins. Oligomerization with wt αA- or αB-crystallins reduces the susceptibility of αA(1-162) to degradation by the UPP. In addition, the presence of αA(1-162) in the hetero-oligomers also affects the degradation of wt αA- and αB-crystallins.