Disruption of heat shock factor 1 reveals an essential role in the ubiquitin proteolytic pathway

Abstract

Inhibition of proteasome-mediated protein degradation machinery is a potent stress stimulus that causes accumulation of ubiquitinated proteins and increased expression of heat shock proteins (Hsps). Hsps play pivotal roles in homeostasis and protection in a cell, through their well-recognized properties as molecular chaperones. The inducible Hsp expression is regulated by the heat shock transcription factors (HSFs). Among mammalian HSFs, HSF1 has been shown to be important for regulation of the heat-induced stress gene expression, whereas the function of HSF2 in stress response is unclear. Recent reports have suggested that both HSF1 and HSF2 are affected during down-regulation of ubiquitin-proteasome pathway (Y. Kawazoe et al., Eur. J. Biochem. 255:356-362, 1998; A. Mathew et al., Mol. Cell. Biol. 18:5091-5098, 1998; D. Kim et al., Biochem. Biophys. Res. Commun. 254:264-268, 1999). To date, however, no unambiguous evidence has been presented as to whether a single specific HSF or multiple members of the HSF family are required for transcriptional induction of heat shock genes when proteasome activity is down-regulated. Therefore, by using loss-of-function and gain-of-function strategies, we investigated the specific roles of mammalian HSFs in regulation of the ubiquitin-proteasome-mediated stress response. Here we demonstrate that HSF1, but not HSF2, is essential and sufficient for up-regulation of Hsp70 expression during down-regulation of the ubiquitin proteolytic pathway. We propose that specificity of HSF1 could be an important therapeutic target during disease pathogenesis associated with abnormal ubiquitin-dependent proteasome function.

FIG. 1

Heat shock response caused by inhibition of the ubiquitin-mediated proteolysis (A) HSF HSE-binding activity in whole cell extracts from control (C), heat-shocked (HS; 42°C for 1 and 6 h), hemin-treated (40 μM for 6, 16, and 24 h), MG132-treated (10 μM for 1, 3, 6, and 8 h), and clasto-lactacystin β-lactone-treated (10 or 5 μM for 3 and 8 h) K562 cells was analyzed by gel mobility shift assay. Extracts (12 μg) were incubated with a ³²P-labeled oligonucleotide representing the proximal HSE of the human hsp70 promoter. Protein-DNA complexes were resolved on a 4% nondenaturing polyacrylamide gel as described elsewhere (26). (B) Transcription rates of hsp70 and hsp90 genes were analyzed by nuclear run-on assay. Equal number of nuclei from control (C), heat-shocked (HS; 1 and 6 h at 42°C), hemin-treated (40 μM for 6, 16, and 24 h), and MG132-treated (10 μM for 1, 3, and 6 h) K562 cells were used for in vitro [³²P]dUTP labeling of newly synthesized transcripts which were hybridized to immobilized DNA probes for human hsp70, human hsp90/89α, rat GAPDH, and a Bluescript vector (BS) as described elsewhere (3). GAPDH was used as an internal control. (C and D) For protein analysis, whole cell extracts (12 μg) isolated from control (C), MG132-treated (10 μM for 3, 8, and 16 h), heat-shocked (HS; 42°C for 1 and 4 h), hemin-treated (40 μM for 8, 16, and 24 h), and clasto-lactacystin β-lactone-treated (10 μM for 3 and 8 h) K562 cells were analyzed by SDS-PAGE and Western immunoblotting using antibodies against HSF1, HSF2, Hsp70, Hsp90, Hsc70, and actin. (E) Poly(A) mRNA from control (C) and MG132-treated (10 μM for 2, 4, 6, and 8 h) K562 cells was analyzed by Northern blotting using ³²P-labeled cDNA probes for human HSF2, hsp70, and GAPDH. GAPDH was used as a control for equal loading. mRNA sizes are indicated on the right.

FIG. 2

Proteasome inhibition induces HSF HSE binding in the absence of HSF2 activity. Whole cell extracts from control (C), heat-shocked (HS; 42°C for 1 h), hemin-treated (He; 40 μM for 16 h), and MG132-treated (MG; 10 μM for 3 h) mock-transfected K562 cells (vector) or K562 cells overexpressing the HSF2-α (2α-C7) or HSF2-β (2β-D5) isoform were analyzed as for Fig. 1A.

FIG. 3

HSF1, but not HSF2, can restore Hsp70 expression. (A) Whole cell extracts from control (C), heat-shocked (HS; 42°C for 1 h without [1] or with [R] a 3-h recovery at 37°C), and MG132-treated (10 μM for 3 and 8 h) wild-type (+/+) and hsf1^−/− MEF cells, as well as wild-type and hsf1^−/− cells transiently transfected with mouse HSF1 (+ HSF1), were analyzed as described for Fig. 1A. (B) The samples described above were analyzed by SDS-PAGE and immunoblotted with antibodies against HSF1, HSF2, Hsp70, Hsp90, Hsc70, and actin. (C) Whole cell extracts from control (C), heat-shocked (HS; 42°C for 1 h without [1] or with [R] a 3-h recovery at 37°C), and MG132-treated (10 μM for 8 h) MEF cells deficient for HSF1, as well as hsf1^−/− cells transiently transfected with mouse HSF2 (+ HSF2) or HSF1 (+ HSF1), were subjected to gel mobility shift analysis (upper panels) and Western immunoblotting (lower panels) using antibodies against HSF2, HSF1, Hsp70, and actin. (D) Whole cell extracts from control (C) and MG132-treated (10 μM for 8 h) hsf1^−/− cells transiently transfected with Flag epitope-tagged mouse HSF1 (+ HSF1) and HSF2 (+ HSF2) were subjected to Western immunoblotting using antibodies against HSF1, HSF2, Flag, Hsp70, and Hsc70.

FIG. 4

Model of HSF1-dependent Hsp70 induction in the ubiquitin (Ub)-proteasome pathway. Down-regulation of ubiquitin-mediated proteolysis by proteasome inhibitors, such as MG132 and clasto-lactacystin β-lactone, results in the accumulation of malfolded, abnormal, and short-lived proteins normally degraded by the 26S proteasome (6). Increase in the amount of polyubiquitinated proteins leads to oligomerization, phosphorylation, and acquisition of HSF1 HSE-binding activity, which in turn induces hsp70 transcription and accumulation of the Hsp70 protein, leading to enhanced molecular chaperone activity. It can be speculated that by associating with the polyubiquitinated proteins, molecular chaperones, among them Hsp70, promote the cellular survival by preventing protein aggregation and facilitating the malfolded proteins to refold. On the other hand, proteasome inhibition results in accumulation and partial oligomerization of the labile HSF2 protein, but the specific role of HSF2 under these conditions remains to be elucidated.