Heat Shock Response and Protein Degradation: Regulation of HSF2 by the Ubiquitin-Proteasome Pathway

Mammalian cells coexpress a family of heat shock factors (HSFs) whose activities are regulated by diverse stress conditions to coordinate the inducible expression of heat shock genes. Distinct from HSF1, which is expressed ubiquitously and activated by heat shock and other stresses that result in the appearance of nonnative proteins, the stress signal for HSF2 has not been identified. HSF2 activity has been associated with development and differentiation, and the activation properties of HSF2 have been characterized in hemin-treated human K562 erythroleukemia cells. Here, we demonstrate that a stress signal for HSF2 activation occurs when the ubiquitin-proteasome pathway is inhibited. HSF2 DNA-binding activity is induced upon exposure of mammalian cells to the proteasome inhibitors hemin, MG132, and lactacystin, and in the mouse ts85 cell line, which carries a temperature sensitivity mutation in the ubiquitin-activating enzyme (E1) upon shift to the nonpermissive temperature. HSF2 is labile, and its activation requires both continued protein synthesis and reduced degradation. The downstream effect of HSF2 activation by proteasome inhibitors is the induction of the same set of heat shock genes that are induced during heat shock by HSF1, thus revealing that HSF2 affords the cell with a novel heat shock gene-regulatory mechanism to respond to changes in the protein-degradative machinery.     

Heat shock gene-expression in HSP-70 and HSF1 gene-transfected human epidermoid A-431 cells

Thermotolerant cells display attenuated heat shock protein 70 kD (HSP-70) gene expression and signal transduction such as intracellular Ca2+ concentration and inositol trisphosphate in response to sublethal heat. To further investigate the regulation of heat shock gene expression, we developed constructs containing human HSP-70 and HSF1 genes and transfected human epidermoid A-431 cells. These cells were chosen because skin cells are especially vulnerable to heat shock and other environmental stressors. We report that A431 cells can be successfully transfected with HSP-70 and HSF1 genes as shown by the elevated levels of respective message and protein. Overexpression of HSP-70 in cells transfected with HSP-70 gene led to a down-regulation of the HSF1 gene expression. Interestingly, transfection of cells with the HSF1 gene was not associated with increased expression of HSP-70. Exposure of HSF1 gene-transfected cells to heat resulted in a transient but significant increase in HSP-70 gene expression as compared to that found in vector-transfected cells, which was completely inhibited by treatment with staurosporine. In conclusion, we have demonstrated successful transfection of human A-431 cells with HSF1 and HSP-70 genes, where the regulation of their expression can be studied. (Mol Cell Biochem 167: 145-152, 1997)     

Heat-Induced Proteasomic Degradation of HSF1 in Serum-Starved Human Fibroblasts Aging in Vitro

The exposure of human fibroblasts (HF) aging in vitro to heat shock resulted in an attenuated expression of the heat shock-inducible HSP70. When late passage cells were cultured in the continuous presence of serum, we observed a reduced accumulation of the cytoplasmic polyadenylated HSP70 mRNA. The levels of HSF1 activation and nuclear HSP70 mRNA were comparable to those of early passage cells (M. A. Bonelli et al., Exp. Cell Res. 252, 20-32, 1999). When late passage cells were serum-starved overnight, we observed a reduced activation of HSF1 and a decreased level of HSP70 mRNA during heat shock. However, at 37 degrees C the levels of HSF1 differed little between late passage HF and early passage cells, irrespective of the presence of serum. Interestingly, during heat shock a marked decrease in the level and, consequently, in the binding activity of HSF1 was noted only in serum-starved, late passage HF. The decrease in the level of HSF1 was counteracted by back addition of serum to the cells during heat shock. Addition of the specific proteasome inhibitor MG132 blocked a decrease in HSF1 during heat shock, maintaining levels observed in late passage cells and HSF1 activity comparable to that of early passage HF. The recovery of the level and activity of HSF1 observed in late passage HF incubated in the presence of MG132 suggests that heat shock unmasks a latent proteasome activity responsible for HSF1 degradation.