Abstract
Purpose As far as we know, during the development of age-related macular degeneration (AMD), the activity of proteasome in retinal pigment epithelium cells (RPE) gradually decreases. And a lot of research has shown that age-related macular degeneration is closely related to inflammation and autoimmune. Moreover, there are many cytokines (CKs) involved in the course of inflammation. In this study, we are going to investigate how the decrease of proteasome activity affects the production of interleukin-6 (IL-6) in human retinal pigment epithelium cells (ARPE-19). Methods Cultured ARPE-19 was treated with or without MG132, a proteasome inhibitor, and the levels of IL-6 mRNA (messenger ribonucleic acid) in RPE at 1 h, 4 h, 8 h, and IL-6 protein in the culture medium at 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h were measured by real-time polymerase chain reaction (real-time PCR) and enzyme-linked immunosorbent assay (ELISA). The protein levels of MCP-1 (monocyte chemoattractant protein-1) in the culture medium at 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h were also measured by ELISA. Then we tested which of cell signal pathways regulating the production of IL-6 were activated when we added MG132 into the medium by Western blot and electrophoretic mobility shift assays (EMSA). After that, we put the inhibitors of these activated cell signal pathways into the medium individually to see which inhibitor can counteract the effect of upregulating the levels of IL-6 in the culture medium of RPE. Results MG132 decreased the secretion of MCP-1 in the culture medium of RPE, but it increased the expression of IL-6 mRNA in RPE and IL-6 protein level in the culture medium of RPE. MG132 treatment was also found to enhance the level of phosphorylated p38 mitogen-activated protein kinases (MAPKs) and c-Jun N-terminal Kinase (JNK) by Western blotting. More importantly, the effect of MG132 on upregulating the levels of IL-6 was inhibited by SB203580, an inhibitor of P38 MAP kinases. But the JNK inhibitor, SP600125, cannot prevent the effect of upregulating the levels of IL-6 by MG132 in the RPE culture medium. Conclusions We concluded that the proteasome inhibitor, MG132, upregulates IL-6 production in RPE cells through the activation of P38 MAPKs.
Highlights
Age-related macular degeneration (AMD) is a disease that causes varying degrees of blindness in senior people especially in developed nations [1, 2], and the mechanism of this disease is still unclear
To further investigate the relationship between the inactivity of proteasome and the expression of IL-6 in retinal pigment epithelial (RPE), we evaluated the effect of inhibition of proteasome activity on the production of IL-6 as well as other relevant inflammatory cytokines and its mechanism. e data suggest that the inactivity of proteasome upregulates the IL-6 expression in RPE cells through the activation of the P38 mitogenactivated protein kinases (MAPKs) pathway
To research if P38 MAPKs inhibition will block the activation of AP-1 induced by proteasome inhibition, we cultured the RPE in the presence of MG132 (10 μM) and in the presence or absence of SB203580 (10 μM) at 1, 2, 4, and 8-hour time point (Figure 8). en we found that there were no obvious differences between MG132 group and MG132 plus SB203580 group in the AP-1 DNAbinding activity. erefore, we did not think P38 MAPKs activated by proteasome inhibition would activate AP-1
Summary
Age-related macular degeneration (AMD) is a disease that causes varying degrees of blindness in senior people especially in developed nations [1, 2], and the mechanism of this disease is still unclear. Considerable evidence shows that retinal oxidative stress [3] and inflammation [4] have been documented with strong association with the development of AMD, both of which are partially regulated by the ubiquitin-proteasome pathway (UPP) [5,6,7]. As other kinds of cells, RPE have a normally active UPP [17], Journal of Ophthalmology and the activity of UPP decreases in different human tissues (including skin, muscle, kidney, liver, lung, heart, lentis, and RPE) with the increase of age [17, 31, 32], the relationship between the decline in proteasome activity in RPE and the production of inflammatory cytokine IL-6 which plays an important role in cell growth and inflammatory reactions [30, 33] remains to be obscured. To further investigate the relationship between the inactivity of proteasome and the expression of IL-6 in RPE, we evaluated the effect of inhibition of proteasome activity on the production of IL-6 as well as other relevant inflammatory cytokines and its mechanism. e data suggest that the inactivity of proteasome upregulates the IL-6 expression in RPE cells through the activation of the P38 MAPKs pathway
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