Proteasome inhibition induces <Emphasis Type="Italic">hsp30</Emphasis> and <Emphasis Type="Italic">hsp70</Emphasis> gene expression as well as the acquisition of thermotolerance in <Emphasis Type="Italic">Xenopus laevis</Emphasis> A6 cells

Previous studies have shown that inhibiting the activity of the proteasome leads to the accumulation of damaged or unfolded proteins within the cell. In this study, we report that proteasome inhibitors, lactacystin and carbobenzoxy-l-leucyl-l-leucyl-l-leucinal (MG132), induced the accumulation of ubiquitinated proteins as well as a dose- and time-dependent increase in the relative levels of heat shock protein (HSP)30 and HSP70 and their respective mRNAs in Xenopus laevis A6 kidney epithelial cells. In A6 cells recovering from MG132 exposure, HSP30 and HSP70 levels were still elevated after 24 h but decreased substantially after 48 h. The activation of heat shock factor 1 (HSF1) may be involved in MG132-induced hsp gene expression in A6 cells since KNK437, a HSF1 inhibitor, repressed the accumulation of HSP30 and HSP70. Exposing A6 cells to simultaneous MG132 and mild heat shock enhanced the accumulation of HSP30 and HSP70 to a much greater extent than with each stressor alone. Immunocytochemical studies determined that HSP30 was localized primarily in the cytoplasm of lactacystin- or MG132-treated cells. In some cells treated with higher concentrations of MG132 or lactacystin, we observed in the cortical cytoplasm (1) relatively large HSP30 staining structures, (2) colocalization of actin and HSP30, and (3) cytoplasmic areas that were devoid of HSP30. Lastly, MG132 treatment of A6 cells conferred a state of thermotolerance such that they were able to survive a subsequent thermal challenge.     

Heat shock protein 25 or inducible heat shock protein 70 activates heat shock factor 1: dephosphorylation on serine 307 through inhibition of ERK1/2 phosphorylation

The expression of heat shock proteins (HSPs) is known to be increased via activation of heat shock factor 1 (HSF1), and excess expression of HSPs exerts feedback inhibition of HSF1. However, the molecular mechanism to modulate such relationships between HSPs and HSF1 is not clear. In the present study, we show that stable transfection of either Hsp25 or inducible Hsp70 (Hsp70i) increased expression of endogenous HSPs such as HSP25 and HSP70i through HSF1 activation. However, these phenomena were abolished when the dominant negative Hsf1 mutant was transfected to HSP25 or HSP70i overexpressed cells. Moreover, the increased HSF1 activity by either HSP25 or HSP70i was found to result from dephosphorylation of HSF1 on serine 307 that increased the stability of HSF1. Either HSP25 or HSP70i inhibited ERK1/2 phosphorylation because of increased MKP1 phosphorylation by direct interaction of these HSPs with MKP1. Treatment of HOS and NCI-H358 cells, which showed high expressions of endogenous HSF1, with small interfering RNA (siRNA) of either HSP27 (siHSP27)or HSP70i (siHSP70i) inhibited both HSP27 and HSP70i proteins; this was because of increased ERK1/2 phosphorylation and serine phosphorylation of HSF1. The results, therefore, suggested that when the HSF1 protein level was high in cancer cells, excess expression of HSP27 or HSP70i strongly facilitates the expression of HSP proteins through HSF1 activation, resulting in severe radio- or chemoresistance.     

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.     

Higher induction of heat shock protein 72 by heat stress in cisplatin-resistant than in cisplatin-sensitive cancer cells

Induction of the heat shock proteins (HSPs) is involved in the increased resistance to cancer therapies such as chemotherapy and hyperthermia. We used two human ovarian cancer cell lines; a cisplatin (CDDP)-sensitive line A2780 and its CDDP-resistant derivative, A2780CP. The concentration of intracellular glutathione (GSH) is higher (2.7-fold increase) in A2780CP cells than in A2780 cells. A mild treatment with a heat stress (42 degrees C for 30 min) induced synthesis of both the heat shock protein 72 (Hsp72) mRNA and the HSP72 protein in A2780CP cells, but not in A2780 cells. In contrast, a severe heat stress (45 degrees C for 30 min) increased synthesis of the HSP72 protein in the two cell lines. The induced level of the HSP72 protein by the severe treatment was higher in A2780CP than in A2780 cells. The gel mobility shift assay showed that DNA binding activities of the heat shock factor (HSF) in the two cell lines were induced similarly and significantly by the mild heat stress. Immunocytochemistry using an anti HSF1 antibody also indicated that mild heat stress activated the HSF1 translocation from the cytosol to the nucleus similarly in the both cell lines. Pretreatment of CDDP-sensitive A2780 cells with N-acetyl-L-cysteine, a precursor of GSH, effectively enhanced induction of the Hsp72 mRNA by the mild heat stress. The present findings demonstrate that induction of the Hsp72 mRNA by the mild heat stress was more extensive in CDDP-resistant A2780CP cells. It is likely that the higher GSH concentration in A2780CP cells plays an important role in promoting Hsp72 gene expression induced by the mild heat stress probably through processes downstream of activation of HSF-DNA binding.     

Regulatory interfaces between the stress protein response and other gene expression programs in the cell

The stress protein response involves the immediate reprogramming of gene expression in cells exposed to proteomic insult leading to massive synthesis of heat shock proteins (HSP). We have examined the outcome when cells are induced to activate two other gene expression programs--the acute inflammatory response and entry of quiescent cells into the cell cycle--and then exposed to protein stress. We find that these responses are mutually antagonistic with, on the one hand, heat shock factor 1 (HSF1) inhibition through the phosphorylation of inhibitory serine residues after inflammatory or mitogenic stimulus and, on the other hand, after stress, HSF1 directly repressing the promoters of genes that mediate acute inflammation and mitogenesis. The expression of the stress protein response during periods of acute protein damage was shown to lead to efficient activation of HSF1 and HSP expression accompanied by repression of other gene expression programs.