The ubiquitin-proteasome pathway (UPP) plays an important role in regulating gene expression. upon impairment of the UPP. These data suggest that impairment of the UPP in RPE may be one of the causes of retinal inflammation and abnormal functions of monocyte and the complement system during the pathogenesis of age-related macular degeneration. for 14 days. The cells were then transferred to PBS and exposed to blue light for 15 min. Cells that accumulated … To determine whether A2E-mediated photooxidation also alters the secretion of these inflammation-related factors we determined the levels of these factors in the medium. As shown in Fig. 31.2 exposure of A2E-containing RPE to blue light resulted in a 20 % and >2-fold increase in levels of IL-6 and IL-8 and a 70-80 % decrease in levels of MCP-1 and CFH in the medium. SNX-2112 Accumulation of A2E alone increased the levels of IL-6 IL-8 MCP-1 and CFH marginally (Fig. 31.2) whereas exposure to blue light alone had no significant effect on levels of these inflammation- related factors in the medium. Fig. 31.2 A2E-mediated photooxidation alters the secretion of inflammation related factors. Confluent cultured ARPE-19 cells were loaded with 10 μM for 14 days. The cells were then transferred to PBS and exposed to blue light for 10 min. Cells that … 31.3 Photooxidation impairs the function of SNX-2112 the UPP Our previous work showed that the proteasome is the most sensitive component of the UPP to oxidative inactivation [47 HIP 48 It is also known that the UPP is involved in regulating gene expressions by controlling signaling pathways and levels of transcript factors. It is plausible that the photooxidation induced changes in expression and secretion of the inflammation-related factors were related to the impairment of the UPP. To confirm previous SNX-2112 results that physiologically relevant levels of oxidative stress SNX-2112 inactivate SNX-2112 the proteasome we determine the effects of A2E-mediated photooxidation on the chymotrypsin-like and trypsin-like activities of the proteasome. As shown in Fig. 31.3 exposure of A2E-containing RPE to blue light resulted in a 70-80 % decrease in trypsin-like and chymotrypsin-like peptidase activities of the proteasome. Accumulation of A2E alone or exposure to blue light alone had no detectible difference in these peptidase activities of the proteasome. This data confirmed our previous results that the proteasome is a sensitive target of oxidative insults. Fig. 31.3 A2E-mediated photooxidation inactivates the proteasome in cultured RPE. Confluent cultured ARPE-19 cells were loaded with 10 μM for 14 days. The cells were then transferred to PBS and exposed to blue light for 15 min and harvested. Cells that … 31.3 Chemical Inhibition of the Proteasome in RPE Results in Similar Changes to that Caused by Photooxidation in Expression and Secretion of Inflammation-Related Factors To test the hypothesis that photooxidation alters the expression and secretion of inflammation-related factors via impairment of the UPP we inhibited proteasome activity in RPE by MG132 and determined the expression and secretion of these inflammation-related factors. We found that inhibition of proteasome resulted in a dramatic increase in mRNA levels for IL-6 and SNX-2112 IL-8 (Fig. 31.4a b). Levels of mRNA for IL-8 increased over 50-fold upon inhibition of the proteasome (Fig. 31.4b). Similar to photooxidation proteasome inhibition resulted in a 70-80 % decrease in levels of mRNA for MCP-1 and CFH (Fig. 31.4c d). Fig. 31.4 Inhibition of the proteasome alters the expression of inflammation-related genes. Confluent cultured ARPE-19 cells were incubated in fresh medium in the absence or presence of 10 μM for 8 h. Levels of for … To determine whether proteasome inhibition also alter the secretion of these inflammation-related factors we determined the levels of these factors in the medium. As shown in Fig. 31.5 inhibition of the proteasome only marginally increased the secretion of IL-6 (Fig. 31.5a) but increased the secretion of IL-8 by >2-fold (Fig. 31.5). Consistent with the decrease in mRNA levels protein levels of MCP-1 and CFH in the medium decreased 80-90 % when the proteasome in RPE was inhibited (Fig. 31.5c d). These data demonstrate that impairment of the UPP.