Up–regulation of heat–shock protein expression by proteasome inhibition: A possible anti–apoptotic mechanism

Abstract

Abstract: : Purpose: Our previous studies have shown that interferon–γ (IFN–γ) induces apoptosis of lens epithelial cells (LECs) and proteasome inhibition protects the cells from IFN–γ induced apoptosis. Anti–apoptotic activity of heat shock proteins (HSPs) and up–regulation of HSPs by proteasome inhibition have been reported recently. Therefore, we tested the hypothesis that induced expression of HSPs is a mechanism by which proteasome inhibition protects LECs from IFN–γ induced apoptosis. Method: Murine lens epithelial αTN4–1 cells were grown in DMEM containing 10% fetal bovine serum, 2 mM glutamine and 100 µg/ml gentamycin. The cells were treated with 100 U/ml IFN–γ, 10 µM MG132 (proteasome inhibitor) or both, and incubated for 12 hours. mRNA and protein expression were observed by RT–PCR and western blot analysis, respectively. Results: MG132 alone or IFN–γ + MG132 caused a >10 fold increase in HSP27, a small increase (1.2–1.6 fold) in αB–crystallin but no change in HSP70 or HSP 90 mRNA expression, compared to no treatment or IFN–γ alone. At the protein level, a dramatic increase in HSP70, >10 fold increase in HSP27, 3 to 4 fold increase in HSP90 but no significant change in αB–crystallin expression, was observed in MG132 or IFN–γ + MG132 treated cells, compared to no treatment or IFN–γ alone. In contrast, down–regulation of αA–crystallin was observed at both the mRNA and protein levels by MG132 or by IFN–γ + MG132. To elucidate the mechanism of HSP induction at the transcriptional level, we looked at expression levels of lens epithelium derived growth factor (LEDGF) and heat–shock transcription factors (HSFs), HSF1 and HSF2. MG132 caused no significant change in LEDGF mRNA or HSF1 protein levels, but a 3 to 4 fold increase in HSF2 protein expression. Conclusion: Proteasome inhibitor MG132, at a concentration that prevents IFN–γ induced apoptosis, causes up–regulation of HSF2, HSP27, HSP70, HSP90 and down–regulation of αA–crystallin in αTN4–1 cells. These proteins may play a role in prevention of IFN–γ induced apoptosis by proteasome inhibition.

Keywords: cytokines/chemokines • apoptosis/cell death • stress response