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N. Awasthi, B.J. Wagner; Heat Shock Protein Induction Is Required for Proteasome Inhibitor–Mediated Rescue From Apoptosis . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3608.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Our previous studies have shown that proteasome inhibition protects lens epithelial cells (LECs) from IFN–γ induced apoptosis. Antiapoptotic activity of proteasome inhibition correlated with increased expression of HSF2, HSP27, HSP70 and HSP90; decreased expression of αA–crystallin and inhibition of caspase–1, –8 and –6 activities. In this study, we tested the hypothesis that HSP induction is not only correlated with, but is required for this antiapoptotic effect. Methods: Murine lens epithelial αTN4–1 cells were grown in DMEM containing 10% FBS, 2mM glutamine and 100 µg/ml gentamycin. At 75–80% confluency, media was changed and cells were pretreated with 100 µM quercetin (HSP inhibitor) for 24 hours and then treated with 100 U/ml IFN–γ, 10 µM MG132 (proteasome inhibitor) or both. HSP expression was observed by western blot analysis. Caspase activities were measured by cleavage of colorimetric substrates. Apoptosis was measured by phase–contrast microscopy and by flow cytometry using annexin V–FITC and propidium iodide staining. Results: Quercetin, a bioflavonoid, has been reported to affect the stress response by inhibiting the synthesis of HSPs. In our study, quercetin pretreatment inhibited the induction of HSPs and HSFs caused by the proteasome inhibitor, MG132. Inhibition was > 75% for HSP27 and HSP90, 62% for HSP70 and 55% for HSF2. Quercetin pretreatment also significantly prevented the inhibition of caspase–1, –8 and –6 activities caused by MG132. Phase–contrast microscopy showed that quercetin pretreatment significantly inhibited the antiapoptotic activity of MG132 on IFN–γ induced apoptosis of LECs. Additionally, flow cytometric analysis showed that the percentages of apoptotic cells in control and IFN–γ, MG132, IFN–γ+MG132, Quercetin and Quercetin+IFN–γ+MG132 treated samples were 1.7, 13.2, 0.58, 0.63, 1.17 and 8.5 respectively, confirming the requirement of HSPs for the antiapoptotic effect of MG132. Conclusions: Our present study shows that the antiapoptotic effect of proteasome inhibition on IFN–γ induced apoptosis requires the induced expression of HSPs. The HSPs appear to act at least in part by modulating caspase activities.
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