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A proteasome inhibitor-stimulated Nrf1 protein-dependent compensatory increase in proteasome subunit gene expression
Sivaprakasam Balasubramanian1, Santosh Kanade, Bingshe Han
1Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
Abstract:
The polycomb group (PcG) proteins, Bmi-1 and Ezh2, are important epigenetic regulators that enhance skin cancer cell survival. We recently showed that Bmi-1 and Ezh2 protein level is reduced by treatment with the dietary chemopreventive agents, sulforaphane and green tea polyphenol, and that this reduction involves ubiquitination of Bmi-1 and Ezh2, suggesting a key role of the proteasome. In the present study, we observe a surprising outcome that Bmi-1 and Ezh2 levels are reduced by treatment with the proteasome inhibitor, MG132. We show that this is associated with a compensatory increase in the level of mRNA encoding proteasome protein subunits in response to MG132 treatment and an increase in proteasome activity. The increase in proteasome subunit level is associated with increased Nrf1 and Nrf2 level. Moreover, knockdown of Nrf1 attenuates the MG132-dependent increase in proteasome subunit expression and restores Bmi-1 and Ezh2 expression. The MG132-dependent loss of Bmi-1 and Ezh2 is associated with reduced cell proliferation, accumulation of cells in G(2), and increased apoptosis. These effects are attenuated by forced expression of Bmi-1, suggesting that PcG proteins, consistent with a prosurvival action, may antagonize the action of MG132. These studies describe a compensatory Nrf1-dependent, and to a lesser extent Nrf2-dependent, increase in proteasome subunit level in proteasome inhibitor-treated cells and confirm that PcG protein levels are regulated by proteasome activity.
Insights
Proteasome inhibitor MG132 unexpectedly reduces polycomb group (PcG) proteins Bmi-1 and Ezh2. This occurs via a compensatory Nrf1-dependent increase in proteasome activity, highlighting PcG protein regulation by proteasome function.
Area of Science:
- Epigenetics
- Molecular Biology
- Cancer Research
Background:
- Polycomb group (PcG) proteins Bmi-1 and Ezh2 are crucial epigenetic regulators promoting skin cancer cell survival.
- Previous studies indicated sulforaphane and green tea polyphenol reduce Bmi-1 and Ezh2 via ubiquitination and proteasomal degradation.
- The proteasome's role in regulating PcG proteins is established, but its response to direct inhibition was unclear.
Purpose of the Study:
- To investigate the effect of proteasome inhibitor MG132 on Bmi-1 and Ezh2 levels.
- To elucidate the underlying mechanisms, including proteasome compensatory responses and the role of Nrf1/Nrf2.
- To determine the functional consequences of altered PcG protein levels in response to proteasome inhibition.
Main Methods:
- Treatment of cells with proteasome inhibitor MG132.
- Analysis of Bmi-1 and Ezh2 protein and mRNA levels.
- Measurement of proteasome activity and expression of proteasome subunits.
- Investigation of Nrf1 and Nrf2 involvement using knockdown techniques.
- Cell proliferation, cell cycle, and apoptosis assays.
- Assessment of effects with forced Bmi-1 expression.
Main Results:
- MG132 treatment unexpectedly decreased Bmi-1 and Ezh2 protein levels.
- This decrease correlated with increased proteasome activity and compensatory upregulation of proteasome subunit mRNA.
- Nrf1 and Nrf2 levels increased upon MG132 treatment; Nrf1 knockdown abrogated the proteasome response and restored Bmi-1/Ezh2 levels.
- MG132-induced loss of Bmi-1/Ezh2 led to reduced proliferation, G2 cell cycle arrest, and increased apoptosis.
- Forced Bmi-1 expression counteracted MG132's effects, suggesting PcG proteins antagonize MG132.
Conclusions:
- Cells exhibit a compensatory Nrf1-dependent (and Nrf2-dependent) increase in proteasome subunit expression and activity in response to proteasome inhibition.
- Proteasome activity plays a significant role in regulating the levels of PcG proteins Bmi-1 and Ezh2.
- PcG proteins may act as prosurvival factors that antagonize the cytotoxic effects of proteasome inhibitors like MG132.
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