Alice Y.-C. Liu

JNK phosphorylates the HSF1 transcriptional activation domain: Role of JNK in the regulation of the heat shock response

The role of c‐Jun NH2‐terminal kinase (JNK) signaling cascade in the stress‐inducible phosphorylation of heat shock factor 1 (HSF1) was investigated using known agonists and antagonists of JNK. We showed that treatment of HeLa cells with MG132, a proteasome inhibitor and known JNK activator, caused the transcriptional activation domain of HSF1 to be targeted and phosphorylated by JNK2 in vivo. Dose‐response and time course studies of the effects of heat shock and anisomycin treatment showed a close correlation of the activation of JNK and hyperphosphorylation of HSF1. SB203580 inhibited JNK at the 100 μM concentration and significantly reduced the amount of hyperphosphorylated HSF1 upon heat shock or anisomycin treatment. SB203580 and dominant‐negative JNK suppress hsp70 promoter‐driven reporter gene expression selectively at 45°C but not at 42°C heat stress, suggesting that JNK would be preferentially associated with the protective heat shock response against severe heat stress. The possibility that JNK‐mediated phosphorylation of HSF1 may selectively stabilize the HSF1 protein and confers protection to cells under conditions of severe stress is discussed. J. Cell. Biochem. 82: 326–338, 2001.

Heat Shock Response, Heat Shock Transcription Factor and Cell Aging

A characteristic feature of aging is a progressive impairment in the ability to adapt to environmental challenges. The purpose of this article is to review the evidence of an attenuated response to heat and physiological stresses in a number of mammalian aging model systems, including the human diploid fibroblasts in culture, whole animals and animal-derived cells and cell cultures, as well as peripheral blood mononuclear cells obtained from human donors. Analyses of the regulation and function of heat shock factor 1 (HSF1), a transcription factor that mediates the response to heat shock, showed that while the relative abundance of both the hsf1 transcript and the HSF1 protein did not change as a function of age, the responsiveness of HSF1 to heat-induced activation, as measured by its trimerization and ability to bind to the heat shock element consensus sequence, was inversely related to the age of the cells used. Given the fundamentally important role of heat shock proteins (HSPs) in many aspects of protein homeostasis and signal transduction it seems likely that the inability, or compromised ability, of aging cells and organisms to activate HSF1 and produce HSPs in response to stress would contribute to the well-known increase in morbidity and mortality of the aged when challenged.

Riluzole Increases the Amount of Latent HSF1 for an Amplified Heat Shock Response and Cytoprotection

Background Induction of the heat shock response (HSR) and increased expression of the heat shock proteins (HSPs) provide mechanisms to ensure proper protein folding, trafficking, and disposition. The importance of HSPs is underscored by the understanding that protein mis-folding and aggregation contribute centrally to the pathogenesis of neurodegenerative diseases. Methodology/Principal Findings We used a cell-based hsp70-luciferease reporter gene assay system to identify agents that modulate the HSR and show here that clinically relevant concentrations of the FDA-approved ALS drug riluzole significantly increased the heat shock induction of hsp70-luciferse reporter gene. Immuno-Western and -cytochemical analysis of HSF1 show that riluzole increased the amount of cytosolic HSF1 to afford a greater activation of HSF1 upon heat shock. The increased HSF1 contributed centrally to the cytoprotective activity of riluzole as hsf1 gene knockout negated the synergistic activity of riluzole and conditioning heat shock to confer cell survival under oxidative stress. Evidence of a post-transcriptional mechanism for the increase in HSF1 include: quantitation of mRNAhsf1 by RT-PCR showed no effect of either heat shock or riluzole treatment; riluzole also increased the expression of HSF1 from a CMV-promoter; analysis of the turnover of HSF1 by pulse chase and immunoprecipitation show that riluzole slowed the decay of [35S]labeled-HSF1. The effect of riluzole on HSF1 was qualitatively different from that of MG132 and chloroquine, inhibitors of the proteasome and lysosome, respectively, and appeared to involve the chaperone-mediated autophagy pathway as RNAi-mediated knockdown of CMA negated its effect. Conclusion/Significance We show that riluzole increased the amount of HSF1 to amplify the HSR for cytoprotection. Our study provides novel insight into the mechanism that regulates HSF1 turnover, and identifies the degradation of HSF1 as a target for therapeutics intervention.

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.

SIRT1 Knockdown Promotes Neural Differentiation and Attenuates the Heat Shock Response

Neurons have a limited capacity for heat shock protein (HSP) induction and are vulnerable to the pathogenic consequence of protein misfolding and aggregation as seen in age‐related neurodegenerative diseases. Sirtuin 1 (SIRT1), an NAD+‐dependent lysine deacetylase with important biological functions, has been shown to sustain the DNA‐binding state of HSF1 for HSP induction. Here we show that differentiation and maturation of embryonic cortical neurons and N2a neuroprogenitor cells is associated with decreases in SIRT1 expression and heat shock‐dependent induction of HSP70 protein. Tests of a pharmacological activator and an inhibitor of SIRT1 affirm the regulatory role of SIRT1 in HSP70 induction. Protein cross‐linking studies show that nuclear SIRT1 and HSF1 form a co‐migrating high molecular weight complex upon stress. The use of retroviral vectors to manipulate SIRT1 expression in N2a cells show that shRNA‐mediated knock down of SIRT1 causes spontaneous neurite outgrowth coincident with reduced growth rate and decreased induction of hsp70‐reporter gene, whereas SIRT1 over‐expression blocks the induced neural differentiation of N2a cells. Our results suggest that decreased SIRT1 expression is conducive to neuronal differentiation and this decrease contributes to the attenuated induction of HSPs in neurons. J. Cell. Physiol. 229: 1224–1235, 2014.

Neural differentiation and the attenuated heat shock response

Differentiation of neural progenitor cells of neuroblastoma, pheochromocytoma, and surrogate stem cell lineages from a state resembling stem cells to a state resembling neurons is accompanied by a marked attenuation in induction of the heat shock protein 70 promoter driven-luciferase reporter gene, and induction of the reporter gene in primary embryonic neurons from hippocampus, cortex, and spinal cord is lower still when compared to the differentiated cells. Neural specificity of this phenotype is demonstrated by a negative correlation of hsp70-reporter gene expression and neurite extension under various experimental conditions. Analysis of biochemical events involved in induction of the heat shock response (HSR) reveal a blunted activation of HSF1 DNA-binding activity, and decreased induction of the mRNA(hsp70) and the 72 kDa HSP70 protein. Immunocytochemical staining for HSP70 demonstrates a cytoplasmic staining pattern; heat shock greatly increased the HSP70 staining intensity in the undifferentiated cells and less so in the differentiated cells. Vulnerability of the differentiated cells towards the oxidizer, arsenite, and the excitotoxic glutamate/glycine is demonstrated by the dose-dependent cytotoxic effects of these agents on cell viability and activation of caspase 3/7. Importantly, conditioning heat shock as well as increased expression of HSP70 by gene transfer conferred protection against such cytotoxicity. Together, our results show that neural differentiation is associated with a decreased induction of the heat shock response and an increased vulnerability to stress induced pathologies and death.

Dynamic regulation and involvement of the heat shock transcriptional response in arsenic carcinogenesis

The objective of this study is to better define induction of the heat shock response by arsenite, and to evaluation if induction of heat shock proteins (HSPs) contributes to the carcinogenic activity of arsenite. We show here that arsenite is a ubiquitous inducer of the heat shock response in mammalian cells: that it activated heat shock transcription factor 1 (HSF1) DNA‐binding activity, enhanced hsp 70 promoter, and induced hsp70mRNA and synthesis of HSP chaperones. Using a high throughput hsp70 promoter‐luciferase reporter assay, we observed a hormetic dose response where low concentrations of arsenite stimulated and high concentrations inhibited. Further, the response was time‐dependent such that with longer times of incubation, the dose response shifted to the left. The effect of arsenite in inducing the hsp 70‐luciferase reporter absolutely required a functional HSF1 as it was not observed in HSF1 minus cells but re‐instated by expression of HSF1. Consistent with the suggestion that arsenic targets vicinal cysteine‐SH, we showed that dithiothreitol blocked the effect of arsenite. Assays of cell viability and caspase showed that arsenite caused a dose‐dependent increase in cell death by activation of caspase 3/7 and pre‐induction of HSPs blunted these effects. Using anchorage independent cell growth as a late stage tumor promotion assay, we showed that low concentrations of arsenite had a growth promoting effect, which was enhanced by moderate heat shock. Our study provides evidence that induction of the heat shock response is a sensitive biomarker of arsenic exposure and that induction of HSPs likely contributes to the tumor promotion effect of arsenic. J. Cell. Physiol. 207: 562–569, 2006.

Resolution, Detection, and Characterization of Redox Conformers of Human HSF1

We describe here an experimental protocol for the resolution, detection, and quantitation of the reduced and oxidized conformers of human heat shock factor 1 (hHSF1) and report on the effects in vitro and in vivo of redox-active agents on the redox status, structure, and function of hHSF1. We showed that diamide, a reagent that promotes disulfide bond formation, caused a loss of immunorecognition of the monomeric hHSF1 protein in a standard Western blot detection procedure. Modification of the Western blot procedure to include dithiothreitol in the equilibration and transfer buffers after gel electrophoresis allowed for the detection of a compact, intramolecularly disulfide cross-linked oxidized hHSF1 (ox-hHSF1) in the diamide-treated sample. The effect of diamide was blocked by pretreatment with N-ethylmaleimide and was reversed by dithiothreitol added to the sample prior to gel electrophoresis. Incubation with nitrosoglutathione at 42 °C also promoted the conversion of HSF1 to ox-HSF1; at 25 °C, however, nitrosoglutathione was by itself without effect but blocked the formation of ox-hHSF1 in the presence of diamide. The disulfide cross-linked ox-hHSF1 was monomeric and resistant to the in vitro heat-induced trimerization and activation. The possibility that ox-HSF1 may occur in oxidatively stressed cells was evaluated. Treatment of HeLa cells with 2 mm l-buthionine sulfoximine promoted the formation of ox-HSF1 and blocked the heat-induced activation of HSF DNA binding activity. Our result suggests that hHSF1 may have integrated redox chemistry of cysteine sulfhydryl into its functional responses.

Changes in the regulation of heat shock gene expression in neuronal cell differentiation.

Neuronal differentiation of the NG108-15 neuroblastoma–glioma hybrid cells is accompanied by a marked attenuation in the heat shock induction of the Hsp70-firefly luciferase reporter gene activity. Analysis of the amount and activation of heat shock factor 1, induction of mRNAhsp, and the synthesis and accumulation of heat shock proteins (HSPs) in the undifferentiated and differentiated cells suggest a transcriptional mechanism for this attenuation. Concomitant with a decreased induction of the 72-kDa Hsp70 protein in the differentiated cells, there is an increased abundance of the constitutive 73-kDa Hsc70, a protein known to function in vesicle trafficking. Assessment of sensitivity of the undifferentiated and differentiated cells against stress-induced cell death reveals a significantly greater vulnerability of the differentiated cells toward the cytotoxic effects of arsenite and glutamate/glycine. This study shows that changes in regulation of the HSP and HSC proteins are components of the neuronal cell differentiation program and that the attenuated induction of HSPs likely contributes to neuronal vulnerability whereas the increased expression of Hsc70 likely has a role in neural-specific functions.

Heat shock induction of HSP 89 is regulated in cellular aging

Induction of heat shock proteins (HSPs) was evaluated as a function of age of the IMR-90 human diploid fibroblasts. Heat shock (42 degrees C) markedly increased the synthesis of proteins with apparent molecular weights of 98, 89, 72, 50, 42 and 25 KDa, with HSP 89 and 72 being most prominent. This heat shock induction of HSPs was inversely correlated to the population doubling level (PDL) of the cell cultures used. For example, the synthesis rate of HSP 89 increased from a basal heat shock of young cells (PDL 18); in the old cells (PDL 51), the increase was from 1% to a maximum of 4% at 10-12 hrs after initiation of the heat shock. Western blot analysis showed that HSP 89 constituted approximately 2 and 10% of total cellular proteins in control and heat shocked (42 degrees C, 12-24 hrs) young cells; corresponding values for the old cells were 2 and 4.5%, respectively. Northern blot quantitation of the amount of mRNA hybridizable to cDNA probes of HSP 89 provided evidence that this age-dependent decrease in induction of HSP 89 in IMR-90 cells was attributable to a transcriptional/pre-translational mechanism.