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Q. Bian, A. Taylor, F. Shang; Ubiquitin-Dependent Regulation of Stress Response in Human Lens Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3820.
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Up-regulation of heat shock proteins is an important cellular protective mechanism for withstanding and recovering from environmental insults. Celastrol, a triterpenoid compound isolated from Celastraceae, is a potent inducer of heat shock responses. The objective of this work is to investigate the mechanism by which celastrol induces heat shock response in human lens epithelial cells (HLEC).
HLEC (SRA 01/04) were treated with 1 µM and 3 µM celastrol. The mRNA levels of cytoplasmic chaperones were determined by Real-Time PCR. Protein levels for these chaperones and ubiquitin conjugates were determined by Western blotting. Proteasome activity was determined using fluorogenic peptides as substrates. Cytoprotective effects of celastrol were determined by MTS assay upon exposure to H2O2 or L-canavanine.
Treatment of HLEC with celastrol induced expression of a broad spectrum of cytoplasmic molecular chaperones. Specifically, mRNA levels of αB-crystallin, Hsp27 and Hsp70 increased 20-120-fold upon celastrol treatment. Protein levels of Hsp27, Hsp70 and 90 increased ~ 2-fold. Levels of ubiquitin conjugates increased in a dose-dependent manner upon celastrol treatment. Activities of all of the three peptidases of the proteasome decreased 30-50% upon celastrol treatment. Impairment of functions of the ubiquitin-proteasome pathway (UPP) resulted in changes that are similar to those induced by celatrol, indicating that celastrol may induce stress response via inhibition the proteasome. Consistent with the results obtained upon UPP inhibition, celastrol treatment enhanced the toxicity induced by H2O2 and L-canavanine.
The data indicate that celastrol induces stress response via impairing the proteasome and accumulation of ubiquitin conjugates. Since increasing ubiquitin conjugates by other methods, such as overexpression of ubiquitin or CHIP (a ubiquitin ligase) also induces stress response in HLEC, these data suggest that accumulation of ubiquitin conjugates in cells is a mediator of stress response. The data also indicate that UPP is essential for cells to cope with environmental stress, such as oxidative stress. Thus, age- or stress related impairment of the UPP in the lens may be a contributing factor for the accumulation of damaged proteins and cataractogenesis.
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