Evidence that a rapidly turning over protein, normally degraded by proteasomes, regulates hsp72 gene transcription in HepG2 cells.

Heat shock protein 72/73 (Hsp70) is a cytosolic molecular chaperone that carries out fundamental roles under both normal and stress situations. There is great interest in delineating the mechanisms whereby Hsp70 levels are regulated. We observed that N-acetyl-leucyl-leucyl-norleucinal (ALLN), a synthetic aldehydic tripeptide that inhibits proteasomes, markedly induced Hsp70 levels (up to 30-fold above base line in HepG2 cells and human endothelial cells). Induction of Hsp70 by ALLN was dose-dependent and not related to cell toxicity. ALLN selectively increased Hsp70 levels without affecting Hsp25, Hsp27, Hsp60, Hsp86, Hsp90, Hsp104, or Bip (immunoglobulin heavy chain binding protein) in HepG2 cells. ALLN induced Hsp70 not only by stabilizing the protein but also by dramatically increasing its synthesis. The modulation of Hsp70 synthesis by ALLN resulted from a rapid and marked increase in transcription of the hsp72 gene, since the induction of hsp72 mRNA was blocked in cells co-treated with actinomycin D. hsp72 mRNA levels were affected in a time-dependent manner by exposure to ALLN; significant elevations occurred within 60 min of treatment, and a decline to background levels was observed by 7 h of recovery. The ALLN-induced increase in hsp72 gene expression was associated with trimerization of the heat shock transcriptional factor (HSF1). ALLN did not affect the steady-state level of HSF1 protein. The effects of ALLN appeared to require de novo protein synthesis, since the induction of both HSF1 trimerization and hsp72 transcription was blocked by co-treatment with cycloheximide. When we tested a series of protease inhibitors, only the related aldehydic tripeptides, N-acetyl-leucyl-leucyl-methioninal and the proteasome inhibitor, Cbz-leucyl-leucyl-leucinal, induced Hsp70 levels. The specific proteasome inhibitor, lactacystin, which has a different structure, also induced Hsp70 levels. Overall, our results suggest that a rapidly turning over protein that is normally degraded by proteasomes may be involved in the regulation of Hsp70 synthesis via effects on the hsp70 transcriptional factor, HSF1.