Geldanamycin Restores a Defective Heat Shock Responsein Vivo

Konstanze F Winklhofer,Anja Reintjes,Marius C Hoener,Richard Voellmy,Jörg Tatzelt

doi:10.1074/jbc.m104873200

Abstract

Induced expression of heat shock proteins (Hsps) plays a central role in promoting cellular survival after environmental and physiological stress. We have previously shown that scrapie-infected mouse neuroblastoma (ScN2a) cells fail to induce the expression of Hsp72 and Hsp28 after various stress conditions. Here we present evidence that this impaired stress response is due to an altered regulation of HSF1 activity. Upon stress in ScN2a cells, HSF1 was converted into hyperphosphorylated trimers but failed to acquire transactivation competence. A kinetic analysis of HSF1 activation revealed that in ScN2a cells trimer formation after stress was efficient, but disassembly of trimers proceeded much faster than in the uninfected cell line. Geldanamycin, a Hsp90-binding drug, significantly delayed disassembly of HSF1 trimers after a heat shock and restored stress-induced expression of Hsp72 in ScN2a cells. Heat-induced Hsp72 expression required geldanamycin to be present; following removal of the drug ScN2a cells again lost their ability to mount a stress response. Thus, our studies show that a defective stress response can be pharmacologically restored and suggest that the HSF1 deactivation pathway may play an important role in the regulation of Hsp expression.

Highlights

Induced expression of heat shock proteins (Hsps) plays a central role in promoting cellular survival after environmental and physiological stress
It is important to note that the deregulated stress response in ScN2a cells was restricted to the two most highly inducible stress proteins Hsp[72] and Hsp[28]; exposure of ScN2a cells to various stress conditions resulted in increased expression of Hsp[73], Hsp[90], grp[75], grp78(BiP), and grp[94], as determined by two-dimensional gel electrophoresis
The studies reported here indicate that the defective stress response in scrapie-infected neuroblastoma (ScN2a) cells is due to an impairment of heat shock transcription factor 1 (HSF1) function and can be restored by the Hsp90-binding drug geldanamycin

Summary

The abbreviations used are

Heat shock protein; HSF, heat shock transcription factor; HSE, heat shock element; PK, proteinase K; PBS, phosphate-buffered saline; wt, wild type; PrPC, cellular prion protein; PrPSc, scrapie prion protein. Aged cells exhibit a decreased ability to induce Hsp[72] in response to stress (13) This compromised induction of Hsp expression may reflect an adaptive cellular response. In response to stress HSF1 assembles into homotrimers, binds to specific heat shock element (HSE) sequences present within inducible Hsp genes, and becomes hyperphosphorylated Repression of the last step of HSF1 activation that leads to transactivation-competent factor appears to be mediated through a regulatory domain defined by Green et al (30) and Zuo et al (24). This final regulation step is not yet understood, it appears to involve chaperone interactions (28, 31, 32) as well as phosphorylation/dephosphorylation events (33–38). We demonstrate that the Hsp90-binding drug geldanamycin corrects the defective regulation of HSF1 activity after heat stress in ScN2a cells

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION