Upregulation of heat shock protein expression by proteasome inhibition: an antiapoptotic mechanism in the lens.
OBJECTIVE
Studies have shown that proteasome inhibition protects lens epithelial cells (LECs) against interferon (IFN)-gamma-induced apoptosis. The present study was conducted to test the hypothesis that proteasome inhibition can protect lens cells against apoptosis by upregulating heat shock protein (HSP) expression.


METHODS
Murine lens epithelial alphaTN4-1 cells were treated with combinations of 100 U/mL IFN-gamma, 10 muM MG132 (proteasome inhibitor), and 100 muM quercetin (HSP inhibitor). mRNA and protein expression were observed by RT-PCR and Western blot analysis, respectively. Caspase activities were measured by using cleavage of colorimetric substrate. Apoptosis was measured by phase-contrast microscopy and flow cytometry.


RESULTS
At the mRNA level, the proteasome inhibitor, MG132, caused a >10-fold increase in HSP27 and a small increase (1.2- to 1.6-fold) in alphaB-crystallin but no change in HSP70 or -90. At the protein level, a more than twofold increase in HSP27 and -90, a marked increase in HSP70, but no significant change in alphaB-crystallin, was observed. Downregulation of alphaA-crystallin by MG132 was observed at both the mRNA and protein levels. MG132 caused no significant change in heat shock factor (HSF)-1, but a more than twofold increase in HSF2 and -4 protein expression. MG132 prevented the IFN-gamma-induced increase in caspase-1, -6, and -8 activities. Quercetin decreased MG132-induced expression of HSP27, -70, and -90 by more than 70%, and heat shock factors HSF2 and -4 by more than 65%. Quercetin pretreatment significantly reversed the decrease in caspase-1, -6, and -8 activities and the antiapoptotic effect of MG132 on IFN-gamma-treated LECs.


CONCLUSIONS
The antiapoptotic effect of proteasome inhibition of IFN-gamma-induced apoptosis in LECs correlates with increased expression of HSPs and inhibition of caspase activities. Inhibition of HSP expression restores caspase activities and abolishes the antiapoptotic effect of proteasome inhibition, implicating HSPs as mediators of the protective effect of proteasome inhibition.
Awasthi
Niranjan
N
Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey, Newark, 07101, USA.
Wagner
B J
BJ
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D004338:Drug Combinations; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D005434:Flow Cytometry; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D007371:Interferon-gamma / Q000494:pharmacology*; D007908:Lens, Crystalline / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D007976:Leupeptins / Q000494:pharmacology; D051379:Mice; D008858:Microscopy, Phase-Contrast; D061988:Proteasome Inhibitors; D011794:Quercetin / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015854:Up-Regulation; D038202:alpha-Crystallin A Chain / Q000378:metabolism
D015853:Cysteine Proteinase Inhibitors; D004338:Drug Combinations; D006360:Heat-Shock Proteins; D007976:Leupeptins; D061988:Proteasome Inhibitors; D012333:RNA, Messenger; D038202:alpha-Crystallin A Chain; D007371:Interferon-gamma; D011794:Quercetin; D020169:Caspases; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde