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.     

Neuroprotective drug riluzole amplifies the heat shock factor 1 (HSF1)- and glutamate transporter 1 (GLT1)-dependent cytoprotective mechanisms for neuronal survival

Heat shock factor 1 (HSF1) mediates the cellular response to stress to increase the production of heat shock protein (HSP) chaperones for proper protein folding, trafficking, and degradation; failure of this homeostatic mechanism likely contributes to neurodegeneration. We show that the neuroprotective drug riluzole increased the amount of HSF1 in NG108-15 neuroprogenitor cells by slowing the specific turnover of HSF1 and supporting a more robust and sustained activation of HSF1. Using Hsp70-luciferase as a functional readout of the activity of HSF1, we show that riluzole amplified the heat shock induction of the reporter gene with an optimal increase at 1 μM. Immunocytochemical staining and Western blot quantitation of HSP70 in NG108-15 neuroprogenitor cells and embryonic spinal cord neurons provided corroborative evidence that riluzole amplified the HSF1-dependent regulation of HSP70 expression. Parallel studies on the GLT1 glutamate transporter showed that riluzole increased GLT1-reporter and GLT1 protein expression and that the increase was enhanced by heat shock and coincident with the increased expression of HSP70 and HSP90. This result is consistent with the anti-glutamatergic profile of riluzole and the presence of multiple heat shock elements on the GLT1 gene promoter, suggesting that riluzole may modulate GLT1 expression through HSF1. The increased HSP chaperones and GLT1 transporter blunted glutamate-induced and N-methyl D-aspartate receptor-mediated excitotoxic death. In summary, we show that riluzole increased the amount and activity of HSF1 to boost the expression of HSPs and GLT1 for neuroprotection under stress.     

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.     

Implication of a small GTPase Rac1 in the activation of c-Jun N-terminal kinase and heat shock factor in response to heat shock

Heat shock induces c-Jun N-terminal kinase (JNK) activation as well as heat shock protein (HSP) expression through activation of the heat shock factor (HSF), but its signal pathway is not clearly understood. Since a small GTPase Rac1 has been suggested to participate in the cellular response to stresses, we examined whether Rac1 is involved in the heat shock response. Here we show that moderate heat shock (39-41 degrees C) induces membrane translocation of Rac1 and membrane ruffling in a Rac1-dependent manner. In addition, Rac1N17, a dominant negative mutant of Rac1, significantly inhibited JNK activation by heat shock. Since Rac1V12 was able to activate JNK, it is suggested that heat shock may activate JNK via Rac1. Similar inhibition by Rac1N17 of HSF activation in response to heat shock was observed. However, inhibitory effects of Rac1N17 on heat shock-induced JNK and HSF activation were reduced as the heat shock temperature increased. Rac1N17 also inhibited HSF activation by l-azetidine-2-carboxylic acid, a proline analog, and heavy metals (CdCl)), suggesting that Rac1 may be linked to HSF activation by denaturation of polypeptides in response to various proteotoxic stresses. However, Rac1N17 did not prevent phosphorylation of HSF1 in response to these proteotoxic stresses. Interestingly, a constitutively active mutant Rac1V12 did not activate the HSF. Therefore, Rac1 activation may be necessary, but not sufficient, for heat shock-inducible HSF activation and HSP expression, or otherwise a signal pathway(s) involving Rac1 may be indirectly involved in the HSF activation. In sum, we suggest that Rac1 may play a critical role(s) in several aspects of the heat shock response.     

HSP70 deficiency results in activation of c-Jun N-terminal Kinase, extracellular signal-regulated kinase, and caspase-3 in hyperosmolarity-induced apoptosis

In this study we examined the function of heat shock protein 70 (HSP70) in the hyperosmolarity-induced apoptotic pathway using hsp70.1-/-mouse embryonic fibroblasts (MEFs). When the cells were exposed to hyperosmotic stress, an absence of HSP70 negatively affected cell viability. Caspase-9 and caspase-3 were rapidly activated, and extensive cleavage occurred in focal adhesion and cytoskeletal molecules in the hsp70.1-/-MEFs. In contrast, hsp70.1+/+ MEFs exhibited no caspase-9 or caspase-3 activation and finally recovered intact cell morphology when cells were shifted back to an isosmotic state. Because HSP70 might be involved in the regulation of mitogen-activated protein kinase (MAPK) activities with regard to various cellular activities, we also monitored MAPK phosphorylation. The absence of HSP70 affected c-Jun N-terminal kinase phosphorylation. However, it had no effect on p38. Sustained phosphorylation of extracellular signal-regulated kinase (ERK) was observed during the hyperosmolarity-induced apoptosis of hsp70.1-/-MEFs. Inhibition of ERK activity by the treatment of PD98059 accelerated the apoptotic pathway. ERK phosphorylation was precisely correlated with shift of mitogen-activated protein kinase phosphatase-3 from the soluble to insoluble fraction. Our results demonstrate that the inhibitory effect of HSP70 on caspase-3 activation is sufficient to inhibit apoptosis and that HSP70 exhibits regulatory functions to c-Jun N-terminal kinase and ERK phosphorylation in hyperosmolarity-induced apoptosis.     

Heat shock factor 1 is a key regulator of the stress response in Chlamydomonas

We report here on the characterization of heat shock factor 1 (HSF1), encoded by one of two HSF genes identified in the genome of Chlamydomonas reinhardtii. Chlamydomonas HSF1 shares features characteristic of class A HSFs of higher plants. HSF1 is weakly expressed under non-stress conditions and rapidly induced by heat shock. Heat shock also resulted in hyperphosphorylation of HSF1, and the extent of phosphorylation correlated with the degree of induction of heat shock genes, suggesting a role for phosphorylation in HSF1 activation. HSF1, like HSFs in yeasts, forms high-molecular-weight complexes, presumably trimers, under non-stress, stress and recovery conditions. Immunoprecipitation of HSF1 under these conditions led to the identification of cytosolic HSP70A as a protein constitutively interacting with HSF1. Strains in which HSF1 was strongly under-expressed by RNAi were highly sensitive to heat stress. 14C-labelling of nuclear-encoded proteins under heat stress revealed that synthesis of members of the HSP100, HSP90, HSP70, HSP60 and small HSP families in the HSF1-RNAi strains was dramatically reduced or completely abolished. This correlated with a complete loss of HSP gene induction at the RNA level. These data suggest that HSF1 is a key regulator of the stress response in Chlamydomonas.