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A. F. Fernandes, J. Zhou, J. Sparrow, A. Taylor, P. Pereira, F. Shang; Oxidative Stress Inactivates the Proteasome and Decreases the Expression of Matrix Metalloproteinases (MMPs) and Their Inhibitors (TIMPs) in RPE. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5050.
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Thickening and fracture of Bruch’s membrane are typical features of AMD. The imbalance between MMPs and TIMPs appears to contribute for these phenotypes. The ubiquitin-proteasome pathway (UPP) plays critical roles in regulating several transcription factors and signaling pathways that may affect expression of MMPs and TIMPs. The objective of this study was to determine the role of the UPP in controlling signaling pathways that may affect the expression and secretion of MMPs and TIMPs from cultured RPE cells.
ARPE-19 cells that accumulated A2E were exposed to blue light. In parallel experiments, ARPE-19 cells were treated with or without proteasome inhibitors, 10 µM MG132 or 5 µM epoxomicin, for 2-8 hours. Levels of mRNA for MMP-2, MMP-9, TIMP-1 and TIMP-2 were assessed by real-time RT-PCR. Protein levels of TIMP-1 and 2 in the medium were determined by ELISA. Proteasome activity was determined using a fluorogenic peptide as substrate. Gelatin zymography was used to determine MMP-2 activity.
A2E-mediated photooxidation resulted in a decrease in TIMP-1 and 2, both at RNA and protein levels as well as in proteasome inhibition. Furthermore, MMP-2 was activated upon photooxidation. The expression and secretion of TIMP-1 and 2 were also substantially attenuated when the proteasome was inhibited. The levels of pro-MMP-2 decreased upon proteasome inhibition, and the active form of MMP-2 was not detectable in the medium.
These data demonstrate that the UPP in RPE is a target of oxidative stress and that the UPP plays an important role in regulating the expression of MMPs and TIMPs. Oxidative impairment of the UPP may disrupt the balance between MMPs and TIMPs and contribute to the age-related abnormalities of Bruch’s membrane. Age-related impairment of the UPP by oxidative stress may contribute to the pathogenesis of AMD.
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