Heat Shock Factor-1 DNA Binding Research Articles

Heat shock factor 1 (HSF1) specifically potentiates c-MYC-mediated transcription independently of the canonical heat shock response

Despite its pivotal roles in biology, how the transcriptional activity of c-MYC is tuned quantitatively remains poorly defined. Here, we show that heat shock factor 1 (HSF1), the master transcriptional regulator of the heat shock response, acts as a prime modifier of the c-MYC-mediated transcription. HSF1 deficiency diminishes c-MYC DNA binding and dampens its transcriptional activity genome wide. Mechanistically, c-MYC, MAX, and HSF1 assemble into a transcription factor complex on genomic DNAs, and surprisingly, the DNA binding of HSF1 is dispensable. Instead, HSF1 physically recruits the histone acetyltransferase general control nonderepressible 5 (GCN5), promoting histone acetylation and augmenting c-MYC transcriptional activity. Thus, we find that HSF1 specifically potentiates the c-MYC-mediated transcription, discrete from its canonical role in countering proteotoxic stress. Importantly, this mechanism of action engenders two distinct c-MYC activation states, primary and advanced, which may be important to accommodate diverse physiological and pathological conditions.

Loss of heat shock factor 1 promotes hepatic stellate cell activation and drives liver fibrosis.

Liver fibrosis is an aberrant wound healing response that results from chronic injury and is mediated by hepatocellular death and activation of hepatic stellate cells (HSCs). While induction of oxidative stress is well established in fibrotic livers, there is limited information on stress‐mediated mechanisms of HSC activation. Cellular stress triggers an adaptive defense mechanism via master protein homeostasis regulator, heat shock factor 1 (HSF1), which induces heat shock proteins to respond to proteotoxic stress. Although the importance of HSF1 in restoring cellular homeostasis is well‐established, its potential role in liver fibrosis is unknown. Here, we show that HSF1 messenger RNA is induced in human cirrhotic and murine fibrotic livers. Hepatocytes exhibit nuclear HSF1, whereas stellate cells expressing alpha smooth muscle actin do not express nuclear HSF1 in human cirrhosis. Interestingly, despite nuclear HSF1, murine fibrotic livers did not show induction of HSF1 DNA binding activity compared with controls. HSF1‐deficient mice exhibit augmented HSC activation and fibrosis despite limited pro‐inflammatory cytokine response and display delayed fibrosis resolution. Stellate cell and hepatocyte‐specific HSF1 knockout mice exhibit higher induction of profibrogenic response, suggesting an important role for HSF1 in HSC activation and fibrosis. Stable expression of dominant negative HSF1 promotes fibrogenic activation of HSCs. Overactivation of HSF1 decreased phosphorylation of JNK and prevented HSC activation, supporting a protective role for HSF1. Our findings identify an unconventional role for HSF1 in liver fibrosis. Conclusion: Our results show that deficiency of HSF1 is associated with exacerbated HSC activation promoting liver fibrosis, whereas activation of HSF1 prevents profibrogenic HSC activation.

Inhibition of Translation Initiation and Heat Shock Factor 1 (HSF1): A Novel Strategy Targeting FLT3 Double Mutant and Non-FLT3 Mutant AML Progenitor/Stem Cells

The transcription factor Heat shock factor 1 (HSF1) is a key sensor of proteotoxic stress and plays a major role in cancer biology. In various tumors, downregulation of HSF1 inhibits growth and induces cell death, and its activation was reported to be an adverse prognostic factor in breast and lung cancers. We have reported that inhibition of translation represses DNA-binding of HSF1 in cancers; large scale drug screening identified the eIF4a antagonist rohinitib (RHT) as a potent inhibitor of the HSF1 activation state (Santagata et al, Science 2013). Review of public databases provided us with additional rationale for targeting HSF1 and eIF4a in acute myeloid leukemia (AML): 1) mRNA levels of HSPA8, a primary HSF1 target, are correlated with poor prognosis in AML; 2) eIF4a mRNA levels are highest in AML among 12 cancer types; and 3) Gene set enrichment analysis using microarray dataset of functionally-defined leukemia stem cells (LSCs) and non-LSCs (Nature, Ng et al), based on intra-patient comparisons, reveals that a gene set for translation initiation is highly enriched in LSCs (NES 2.73, FDR = 0.000, p= 0.000).RHT induced marked apoptogenic effects in seven human AML cell lines in culture at low nanomolar concentrations (LD50s; 9.5 to 99.5 nM, IC50s; 4.7 to 8.8 nM, based on AnnexinV/PI-positivity at 72hr). RHT inhibited HSF1 transcriptional activity, as shown by 70% reduction in HSPA8 mRNA levels, and 50% reduction in a luciferase reporter driven by the HSPA1A or HSPA6 promoters. We then tested primary samples from 17 AML patients and bone marrow (BM) samples from eight healthy donors. RHT potently induced apoptosis in AML cells, while relatively sparing normal BM cells (%apoptosis: 65.4% vs 6.67%, P < 0.0001). Importantly, a similarly significant difference in sensitivity was also observed between AML and normal stem/progenitor cell populations (CD45+CD34+CD38-). We further found that the activity of RHT was significantly higher in FLT3-ITD than in FLT3-wt AMLs. Comparing isogenic versions of the cell line Ba/F3, RHT more potently killed FLT3-ITD cells than wild-type (wt) cells (LD50s; 65.3 vs 20.1 nM). Immunoblot analysis showed higher activation-associated phospho-HSF1 (Serine 326) in FLT3-ITD Ba/F3 than in FLT3-wt cells, and growth inhibition by HSF1 knockdown was FLT3-ITD-dependent in those lines, indicating greater dependence of FLT3-ITD cells on HSF1. In an AML xenograft model with GFP-luciferase labeled MOLM-13 cells, RHT significantly reduced tumor burden as measured by bioluminescence imaging and reduction in GFP+ leukemic cells peripheral blood (PB) and BM by day 16. Median survival of the treatment group was significantly longer (24 vs 18 days, p < 0.0001).We also found that RHT was synergistic when combined with FLT3 inhibitors of Type I (sorafenib, quizartinib, midostaurin) or Type II (gilteritinib, and E-6201). Immunoblot analysis showed that oncogenic FLT3 activation is associated with increased phosphorylation of HSF1 (S326), suggesting that decreased phosphorylation of the S326 site associated with FLT3 inhibition and diminished DNA-binding by HSF1 driven by eIF4a inhibition combine to promote synergistic lethality. In support of this, HSF1 knockdown sensitized MOLM-13 and FLT3-ITD+ Ba/F3 cells to FLT3 inhibitors. Importantly, similar combinatorial synergistic effects with type II FLT3 inhibitors were observed in cells with dual mutations of FLT3-ITD and FLT3-D835Y/H. This observation has significant clinical relevance because the development of secondary point mutations is a major resistance mechanism against FLT3 inhibitors.Finally, to test the hypothesis that eIF4a inhibition impairs LSC functions, we treated primary patient-derived xenograft AML PDX cells with RHT ex vivo, then injected identical numbers of live cells into recipient NSG mice. Subsequent leukemia engraftment was delayed by one week vs. controls, and human CD45+ cells in PB and BM at 4 weeks post transplantation were significantly reduced (n = 9, 3.30% vs 0.26% in PB, P < 0.0001). This reduced engraftment capacity of RHT-treated PDX AML-LSCs indicates a toxic effect of eIF4a inhibition on AML-LSCs.Collectively, these preclinical findings strongly support the further development of eIF4a inhibitors for the treatment of AML, especially for high-risk FLT3-mutated AML, with additional potential as LSC-targeted therapy. DisclosuresNo relevant conflicts of interest to declare.

Protonation behavior of histidine during HSF1 activation by physiological acidification.

The expression of eukaryotic molecular chaperones (heat shock proteins, HSPs) is triggered in response to a wide range of environmental stresses, including: heat shock, hydrogen peroxide, heavy metal, low-pH, or virus infection. Biochemical and genetic studies have clearly shown the fundamental roles of heat shock factor 1 (HSF1) in stress-inducible HSP gene expression, resistance to stress-induced cell death, carcinogenesis, and other biological phenomena. Previous studies show that acidic pH changes within the physiological range directly activate the HSF1 function in vitro. However, the detailed mechanism is unclear. Though computational pKa-predications of the amino acid side-chain, acidic-pH induced protonation of a histidine residue was found to be most-likely involved in this process. The histidine 83 (His83) residue, which could be protonated by mild decrease in pH, causes mild acidic-induced HSF1 activation (including in-vitro trimerization, DNA binding, in-vivo nuclear accumulation, and HSPs expression). His83, which is located in the loop region of the HSF1 DNA binding domain, was suggested to enhance the intermolecular force with Arginine 79, which helps HSF1 form a DNA-binding competent. Therefore, low-pH-induced activation of HSF1 by the protonation of histidine can help us better to understand the HSF1 mechanism and develop more therapeutic applications (particularly in cancer therapy). J. Cell. Biochem. 116: 977-984, 2015. © 2015 Wiley Periodicals, Inc.

Inhibiting heat shock factor 1 in human cancer cells with a potent RNA aptamer.

Heat shock factor 1 (HSF1) is a master regulator that coordinates chaperone protein expression to enhance cellular survival in the face of heat stress. In cancer cells, HSF1 drives a transcriptional program distinct from heat shock to promote metastasis and cell survival. Its strong association with the malignant phenotype implies that HSF1 antagonists may have general and effective utilities in cancer therapy. For this purpose, we had identified an avid RNA aptamer for HSF1 that is portable among different model organisms. Extending our previous work in yeast and Drosophila, here we report the activity of this aptamer in human cancer cell lines. When delivered into cells using a synthetic gene and strong promoter, this aptamer was able to prevent HSF1 from binding to its DNA regulation elements. At the cellular level, expression of this aptamer induced apoptosis and abolished the colony-forming capability of cancer cells. At the molecular level, it reduced chaperones and attenuated the activation of the MAPK signaling pathway. Collectively, these data demonstrate the advantage of aptamers in drug target validation and support the hypothesis that HSF1 DNA binding activity is a potential target for controlling oncogenic transformation and neoplastic growth.

Glutamine-mediated Dual Regulation of Heat Shock Transcription Factor-1 Activation and Expression

Regulation of transcriptional activity of heat shock factor-1 (HSF1) is widely thought to be the main point of control for heat shock protein (Hsp) expression. Glutamine increases Hsf1 gene transcription in a C/EBPβ-dependent manner and up-regulates HSF1 activity. Glutamine is an activator for both HSF1 expression and transactivation. Glutamine-induced HSF1 expression provides a novel mechanistic frame for HSF1-Hsp axis regulation. Heat shock transcription factor-1 (HSF1) is the master regulator for cytoprotective heat shock protein (Hsp) expression. It is widely thought that HSF1 expression is non-inducible, and thus the key control point of Hsp expression is regulation of the transactivation activity of HSF1. How HSF1 expression is regulated remains unknown. Herein we demonstrate that glutamine (Gln), a preferred fuel substrate for the gut, enhanced Hsp expression both in rat colonic epithelium in vivo and in cultured non-transformed young adult mouse colonic epithelial cells. This was associated with up-regulation of the transactivation activity of HSF1 via increased HSF1 trimerization, nuclear localization, DNA binding, and relative abundance of activating phosphorylation at Ser-230 of HSF1. More intriguingly, Gln enhanced HSF1 protein and mRNA expression and Hsf1 gene promoter activity. Within the -281/-200 region of the Hsf1 promoter, deletion of the putative CCAAT enhancer-binding protein (C/EBP) binding site abolished the HSF1 response to Gln. C/EBPβ was further shown to bind to this 82-bp sequence both in vitro and in vivo. Gln availability strikingly altered the ratio of C/EBPβ inhibitory and active isoforms, i.e. liver-enriched inhibitory protein and liver-enriched activating protein. Liver-enriched inhibitory protein and liver-enriched activating protein were further shown to be an independent repressor and activator, respectively, for Hsf1 gene transcription, and the relative abundance of these two C/EBPβ isoforms was demonstrated to determine Hsf1 transcription. We show for the first time that Gln not only enhances the transactivation of HSF1 but also induces Hsf1 expression by activating its transcription in a C/EBPβ-dependent manner.

Inhibition of the Heat Shock Response by PI103 Enhances the Cytotoxicity of Arsenic Trioxide

Heat shock factor 1 (HSF1) is a key regulator of the cytoprotective and anti-apoptotic heat shock response and can be activated by arsenite. Inhibition of HSF1 activation may therefore enhance the cytotoxicity of arsenic trioxide (ATO). We show that ATO induced HSF1 phosphorylation at serine 326 (S326) and induced HSF1-dependent expression of heat shock proteins (HSPs) 27 and 70 in cultured cells. HSF1 significantly reduced cell sensitivity to ATO by reducing apoptosis. Disruption of HSF1 function not only reduced ATO induction of HSP27 and 70 but also enhanced ATO cytotoxicity by elevating apoptosis. These results reveal that HSF1 activation and the resulting induction of HSPs may protect cells from ATO cytotoxicity. The diminished expression of HSPs and hypersensitivity to ATO in cells stably depleted of HSF1 was rescued by ectopic expression of wild-type HSF1 but not an S326A substitution mutant, indicating that phosphorylation at S326 was critical for the protective effect of HSF1. Simultaneous treatment of cells with ATO and PI103, an inhibitor of members of the phosphatidylinositol 3-kinase (PI3K) family, suppressed not only ATO-induced expression of an HSP70 promoter-reporter construct and endogenous HSP70 but also phosphorylation of HSF1 S326. PI103 considerably reduced HSF1 transactivation in ATO-treated cells but had only a limited effect on HSF1 nuclear translocation and DNA binding. Furthermore, PI103 enhanced ATO cytotoxicity in an HSF1-dependent manner. Thus, inhibition of S326 phosphorylation by PI103 blocks the transactivation of HSF1 and may consequently suppress ATO induction of the heat shock response and sensitize cells to ATO.

Fas ligation and tumor necrosis factor alpha activation of murine astrocytes promote heat shock factor-1 activation and heat shock protein expression leading to chemokine induction and cell survival

Death-inducing ligands tumor necrosis factor alpha (TNFα) and Fas ligand (FasL) do not kill cultured astrocytes; instead they induce a variety of chemokines including macrophage-inflammatory protein-1α/CC chemokine ligand 3 (CCL3), monocyte chemoattractant protein-1 (CC CCL-2), macrophage-inflammatory protein-2/CXC chemokine ligand 2 (CXCL2, a murine homologue of interleukin 8), and interferon-induced protein of 10 kDa (CXCL10). Induction is enhanced by protein synthesis inhibition suggesting the existence of endogenous inhibitors. ERK, NF-κB, heat shock factor-1 (HSF-1) and heat shock proteins were examined for their possible roles in signal transduction. Inhibition of ERK activation by PD98059 partially inhibited expression of all but FasL-induced CXCL10. Although inhibition of NF-κB DNA binding inhibited chemokine induction, PD98059 did not inhibit TNFα-induced NF-κB DNA binding suggesting that ERK serves an NF-κB-independent pathway. Heat shock itself induced astrocytic chemokine expression; both TNFα and FasL induced HSF-1 DNA binding and Hsp72 production; and Hsp72-induced chemokine expression. Inhibition of either HSF-1 binding with quercetin or heat shock protein synthesis with KNK437 compromised chemokine induction without compromising cell survival. These data suggest that the induction of heat shock proteins via HSF-1 contribute to the TNFα- and FasL-induced expression of chemokines in astrocytes.

Prostaglandin E2 potentiates heat shock-induced heat shock protein 72 expression in A549 cells

The heat shock (HS) response is an important cytoprotective response comprising the expression of heat shock proteins (HSPs) and orchestrated by the heat/stress-induced transcription factor, heat shock factor-1 (HSF-1). Previous studies suggest that the activation threshold and magnitude of the HS response may be modified by treatment with arachidonic acid (AA). We analyzed the effect of exogenous AA and its metabolites, PGE 2, LTD 4, and 15-HETE on HSF-1-dependent gene expression in A549 human respiratory epithelial-like cells. When added at 1 μM, PGE 2 much more than LTD 4, but not 15-HETE increased activity of a synthetic HSF-1-dependent reporter after HS exposure (42 °C for 2 h), but had no effect in the absence of HS. Exposing A549 cells to HS stimulated the release of PGE 2 and treatment with the cyclooxygenase inhibitor, ibuprofen, reduced HS-induced HSF-1-dependent transcription. PGE 2 increased HS-induced HSP72 mRNA and protein expression but EMSA and Western blot analysis failed to show an effect on HSF-1 DNA binding activity or post-translational modification. In summary, we showed that HS stimulates the generation of PGE 2, which augments the generation of HSPs. The clinical consequences of this pathway have yet to be determined.

Alpha-helix 1 in the DNA-binding domain of heat shock factor 1 regulates its heat-induced trimerization and DNA-binding

Heat shock factor 1 (HSF1) primarily regulates various cellular stress responses. The role of α-helix1 (H1) in its DNA-binding domain (DBD) during HSF1 activation remains unknown. Here, HSF1 lacking H1 loses its heat-induced activity, suggesting the importance of the latter. Furthermore, the CD spectra and AMBER prediction show that this H1 deficiency does not change the structure of HSF1 monomer, but does impact its heat-induced trimerization. Point mutation showed that Phe18 in H1 interacts with Tyr60, and that Trp23 interacts with Phe104 by an aromatic–aromatic interaction. Thus, the presence of H1 stabilizes the DBD structure, which facilitates the heat-induced trimerization and DNA-binding of HSF1.

Cisplatin abrogates the geldanamycin-induced heat shock response

Benzoquinone ansamycin antibiotics such as geldanamycin (GA) bind to the NH(2)-terminal ATP-binding domain of heat shock protein (Hsp) 90 and inhibit its chaperone functions. Despite in vitro and in vivo studies indicating promising antitumor activity, derivatives of GA, including 17-allylaminogeldanamycin (17-AAG), have shown little clinical efficacy as single agents. Thus, combination studies of 17-AAG and several cancer chemotherapeutics, including cisplatin (CDDP), have begun. In colony-forming assays, the combination of CDDP and GA or 17-AAG was synergistic and caused increased apoptosis compared with each agent alone. One measurable response that results from treatment with Hsp90-targeted agents is the induction of a heat shock factor-1 (HSF-1) heat shock response. Treatment with GA + CDDP revealed that CDDP suppresses up-regulation of HSF-1 transcription, causing decreased levels of stress-inducible proteins such as Hsp27 and Hsp70. However, CDDP treatment did not prevent trimerization and nuclear localization of HSF-1 but inhibited DNA binding of HSF-1 as shown by chromatin immunoprecipitation. Melphalan, but not camptothecin, caused similar inhibition of GA-induced HSF-1-mediated Hsp70 up-regulation. 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt cell survival assays revealed that deletion of Hsp70 caused increased sensitivity to GA (Hsp70(+/+) IC(50) = 63.7 +/- 14.9 nmol/L and Hsp70(-/-) IC(50) = 4.3 +/- 2.9 nmol/L), which confirmed that a stress response plays a critical role in decreasing GA sensitivity. Our results suggest that the synergy of GA + CDDP is due, in part, to CDDP-mediated abrogation of the heat shock response through inhibition of HSF-1 activity. Clinical modulation of the HSF-1-mediated heat shock response may enhance the efficacy of Hsp90-directed therapy.

Anti-apoptotic Effects of L-Glutamine—Mediated Transcriptional Modulation of the Heat Shock Protein 72 During Heat Shock

During physiologic stress, L-glutamine becomes conditionally essential. Its deficiency results in altered epithelial barrier competence, bacterial translocation, and decreased survival. L-glutamine may attenuate these effects by modulating heat shock protein expression, a well-described effect in vitro. We sought to characterize L-glutamine-dependent transcriptional regulation in heat-shocked intestinal cells and to determine its physiologic relevance. IEC-18 and H4 intestinal cells were used. Heat shock protein 72 (Hsp72) gene expression was determined by Northern blotting and luciferase assays. Heat shock factor-1 (HSF-1) activation was assessed by electromobility shift assay, Western blotting, and HSF-1 minimal promoters. Phosphorylation and trimerization of HSF-1 were determined by immunoprecipitation and native nonreducing gradient polyacrylamide gel electrophoresis (PAGE). Camptothecin-induced apoptosis was monitored using caspase-3 and poly (ADP-ribose) polymerase [PARP]-specific antibodies and DNA Elisa +/- Hsp72 siRNA. L-glutamine specifically augmented Hsp72 transcript abundance and HSF-1 DNA binding during heat shock. No glutamine-dependent differences in HSF-1 phosphorylation, trimerization, nuclear localization during heat shock, or HSF-1 minimal promoter activity were observed. Nevertheless, the presence of L-glutamine was an important determinant of wild-type Hsp72 promoter transcriptional activation. Reduced Hsp72 was associated with increased camptothecin-induced caspase-3 and PARP cleavage in glutamine-deficient cells. siRNA treated cells were less resistant to camptothecin. Taken together, the data suggest that glutamine does not affect the classical pathway of HSF-1 activation and that glutamine-dependent upstream trans -factor binding elsewhere in the Hsp72 promoter or coactivator recruitment may determine Hsp72 abundance. L-glutamine potentiation of Hsp72 is associated with increased epithelial resistance to apoptotic injury.

Differential regulation of activator protein-1 and heat shock factor-1 in myocardial ischemia and reperfusion injury: role of poly(ADP-ribose) polymerase-1.

Poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme activated in response to DNA strand breaks, has been implicated in cell dysfunction in myocardial reperfusion injury. PARP-1 has also been shown to participate in transcription and regulation of gene expression. In this study, we investigated the role of PARP-1 on the signal transduction pathway of activator protein-1 (AP-1) and heat shock factor-1 (HSF-1) in myocardial reperfusion injury. Mice genetically deficient of PARP-1 (PARP-1(-/-) mice) exhibited a significant reduction of myocardial damage after occlusion and reperfusion of the left anterior descending branch of the coronary artery compared with their wild-type littermates. This cardioprotection was associated with a reduction of the phosphorylative activity of JNK and, subsequently, reduction of the DNA binding of the signal transduction factor AP-1. On the contrary, in PARP-1(-/-) mice, DNA binding of HSF-1 was enhanced and was associated with a significant increase of the cardioprotective heat shock protein (HSP)70 compared with wild-type mice. Microarray analysis revealed that expression of several AP-1-dependent genes of proinflammatory mediators and HSPs was altered in PARP-1(-/-) mice. The data indicate that PARP-1 may exert a pathological role in reperfusion injury by functioning as an enhancing factor of AP-1 activation and as a repressing factor of HSF-1 activation and HSP70 expression.

Cholinergic- and stress-induced signaling activities in cells overexpressing wild-type and mutant presenilin-1

This study examined the effects of overexpression of presenilin-1 wild-type (PS1wt) or mutant L286V (PS1m) in human neuroblastoma SH-SY5Y cells on signal transduction systems. Oxotremorine-M-induced activation of AP-1 was 40–53% lower in PS1wt than control cells, and further impaired (63–76%) in PS1m cells. Heat shock (45°C) activated Akt, increased heat shock factor-1 (HSF-1) DNA binding activity, and increased levels of heat shock protein 70, and these responses were not altered by overexpression of PS1wt or PS1m. H 2O 2 also caused a time-dependent increase in HSF-1 DNA binding activity which was similar in all cell lines. Thus, overexpression of PS1wt reduced muscarinic receptor-mediated activation of AP-1, and PS1m overexpression caused greater inhibition, but stress-induced activation of Akt and HSF-1 was unaffected by either PS1wt or PS1m.

Opposing actions of phosphatidylinositol 3-kinase and glycogen synthase kinase-3beta in the regulation of HSF-1 activity.

Elevated temperatures activate the survival promoters Akt and heat shock factor-1 (HSF-1), a transcription factor that induces the expression of heat shock proteins (HSPs), such as HSP-70. Because neuronal mechanisms controlling these responses are not known, these were investigated in human neuroblastoma SH-SY5Y cells. Heat shock (45 degrees C) rapidly activated Akt, extracellular signal-regulated kinases 1 and 2 (ERK1/2), and p38, but only Akt was activated in a phosphatidylinositol 3-kinase (PI-3K)-dependent manner, as the PI-3K inhibitors LY294002 and wortmannin blocked Akt activation, but not ERK1/2 or p38 activation. Akt activation was not blocked by inhibition of p38 or ERK1/2, indicating the independence of these signaling systems. Heat shock treatment also caused a rapid increase in HSF-1 DNA binding activity that was partially dependent on PI-3K activity, as both the PI-3K inhibitors attenuated this response. Because Akt inhibits glycogen synthase kinase-3beta (GSK-3beta), an enzyme that facilitates cell death, we tested if GSK-3beta is a negative regulator of HSF-1 activation. Overexpression of GSK-3beta impaired heat shock-induced activation of HSF-1, and also reduced HSP-70 production, which was partially restored by the GSK-3beta inhibitor lithium. Thus, heat shock-induced activation of PI-3K and the inhibitory effect of GSK-3beta on HSF-1 activation and HSP-70 expression imply that Akt-induced inhibition of GSK-3beta contributes to the activation of HSF-1.

Acute pancreatitis results in induction of heat shock proteins 70 and 27 and heat shock factor-1.

Heat shock proteins (HSPs) 70 and 27 are stress-responsive proteins that are important for cell survival after injury; the expression of these HSPs is regulated primarily by the transcription factor heat shock factor-1 (HSF-1). The purpose of this study was to determine the effect of acute pancreatitis on pancreatic HSPs (70, 27, 60, and 90) expression and to assess potential mechanisms for HSP induction using a murine model of cerulein-induced pancreatitis. We found an increase of both HSP70 and HSP27 levels with expression noted throughout the pancreas after induction of pancreatitis. HSP60 and HSP90 levels were constitutively expressed in the pancreas and did not significantly change with acute pancreatitis. HSF-1 DNA binding activity increased in accordance with increased HSP expression. We conclude that acute pancreatitis results in a marked increase in the expression of HSP70 and HSP27. Furthermore, the induction of HSP70 and HSP27 expression was associated with a concomitant increase in HSF-1 binding activity. The increased expression of both HSP70 and HSP27 noted with pancreatic inflammation may confer a protective effect for the remaining acini after acute pancreatitis.

Rapid activation of heat shock factor-1 DNA binding by H 2O 2 and modulation by glutathione in human neuroblastoma and Alzheimer's disease cybrid cells

Because cellular signaling systems are critical mediators of responses to oxidative stress, a condition associated with neurodegenerative disorders, the redox-dependent regulation of heat shock factor-1 (HSF-1) was investigated in human neuroblastoma SH-SY5Y cells. Exposure of cells to 200 μM H 2O 2 caused a rapid increase in HSF-1 DNA binding that was evident within 10 min, and caused a robust increase that reached levels 8-fold the basal activity. In comparison, the transcription factors, activator protein-1 (AP-1) and early growth response-1 (EGR-1), were activated more slowly and to a lesser extent. Activation of HSF-1 DNA binding activity was associated with a cytosolic to nuclear translocation of HSF-1 protein, and was detected with concentrations of H 2O 2 of 100 μM and greater. Intracellular glutathione modulated H 2O 2-induced HSF-1 DNA binding activity, as depletion of glutathione caused HSF-1 to be activated with lower concentrations of H 2O 2 (25 μM) and supplementation of glutathione blocked HSF-1 activation by 100 to 400 μM H 2O 2. Alzheimer's disease (AD) and control cybrid cells (SH-SY5Y cells in which the mitochondria were replaced with platelet mitochondria from AD or matched control subjects) were used to test the effects of the chronic oxidative stress caused by the excessive production of reactive oxygen intermediates (ROIs) in AD cybrids on HSF-1 activity. Basal and maximal (induced by H 2O 2 in glutathione-depleted cells) HSF-1 DNA binding activity were lower in AD than control cybrids, suggesting that the cells had compensated for excessive ROIs. These results indicate that the activation of HSF-1 is highly sensitive to oxidative stress and is regulated by endogenous antioxidant mechanisms.

Attenuated stress responses in young and old human lymphocytes

Aging generally is understood to be a period defined by altered responses to physiological stress. At the molecular level, several stress responses involving specific gene expression have been revealed, and thermal stress has been tightly linked to induction of the heat shock gene family (D.A. Jurivich. In E. Bittar (ed.), Principles of Medical Biology, Vol. 4, JAI press, San Diego, 1996, pp. 411–462). Perturbations in heat shock gene transcription consistently have been noted in senescent cells from all species examined thus far. Because heat shock proteins serve several vital functions in the immune system, changes in the thermal stress response could potentially contribute to immunosenescence. Inadequate promoter priming by the transactivator of heat shock genes, heat shock factor 1 (HSF1), is thought to account for age-dependent diminution in expression of these genes, although the exact mechanism for this loss is not clearly understood. We have found that human lymphocytes exhibit an age-dependent loss in HSF1–DNA binding, although a range of binding has been observed in both young and old donor cells. This report characterizes a subset of young and old human donor lymphocytes that are non-responders to thermal stress defined by the absence of HSF1–DNA binding after a 42°C heat shock. Whole cell extracts from these donor cells have the capacity to inhibit HSF1–DNA binding when mixed with pre-activated HSF1 from HeLa cells. This inhibitory activity is lost upon heat denaturation and does not appear to be protease mediated. Serial passage of lymphoblasts recapitulates loss of heat inducible HSF1–DNA observed in old donor lymphocytes, thus suggesting that loss of replicative potential and aging lead to altered stress responses. Uncoupling of the thermal response and its potential relevance to apoptosis and aging are discussed.