Heat Shock Transcription Factor 1 Levels Research Articles

HSF1 inhibits microglia activation to attenuate neuroinflammation via regulating miR-214-3p and NFATc2 in Parkinson's disease.

Parkinson's disease (PD) is characterized by microglia activation that leads to neuroinflammation. Heat shock transcription factor 1 (HSF1) is known to exert neuroprotective effects on neurodegenerative diseases. This study sought to analyse the role and mechanism of HSF1 in PD-induced neuroinflammation. The PD mouse models were established using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Animal behaviour capacities and neuronal damage were assessed via behavioural tests, tyrosine hydroxylase (TH) staining, and immunofluorescence. Levels of HSF1, miR-214-3p, nuclear factor of activated T cells 2 (NFATc2), and neuroinflammatory factors were detected via RT-qPCR, Western blotting, and ELISA.Binding relationships between HSF1 and miR-214-3p, miR-214-3p, and NFATc2 were tested via dual-luciferase or chromatin immunoprecipitation assays. Functional rescue experiments were designed to confirm the roles of miR-214-3p and NFATc2. HSF1 expression in brain tissues was downregulated upon MPTP treatment. HSF1 overexpression reduced motor deficits and loss of dopaminergic neurons, increased TH-positive neurons, and repressed neuroinflammation and micro-glia activation. Mechanically, HSF1 bound to the miR-214-3p promoter to increase its expression and inhibited NFATc2 transcription. miR-214-3p downregulation or NFATc2 overexpression reversed the inhibition of HSF1 overexpression on neuroinflammation and microglia activation. Overall, our findings unveiled the therapeutic role of HSF1 in PD-induced neuroinflammation and microglia activation via regulating miR-214-3p and NFATc2.

Heat Shock Factor 1 Directly Regulates Postsynaptic Scaffolding PSD-95 in Aging and Huntington's Disease and Influences Striatal Synaptic Density.

PSD-95 (Dlg4) is an ionotropic glutamate receptor scaffolding protein essential in synapse stability and neurotransmission. PSD-95 levels are reduced during aging and in neurodegenerative diseases like Huntington’s disease (HD), and it is believed to contribute to synaptic dysfunction and behavioral deficits. However, the mechanism responsible for PSD-95 dysregulation under these conditions is unknown. The Heat Shock transcription Factor 1 (HSF1), canonically known for its role in protein homeostasis, is also depleted in both aging and HD. Synaptic protein levels, including PSD-95, are influenced by alterations in HSF1 levels and activity, but the direct regulatory relationship between PSD-95 and HSF1 has yet to be determined. Here, we showed that HSF1 chronic or acute reduction in cell lines and mice decreased PSD-95 expression. Furthermore, Hsf1(+/−) mice had reduced PSD-95 synaptic puncta that paralleled a loss in thalamo-striatal excitatory synapses, an important circuit disrupted early in HD. We demonstrated that HSF1 binds to regulatory elements present in the PSD-95 gene and directly regulates PSD-95 expression. HSF1 DNA-binding on the PSD-95 gene was disrupted in an age-dependent manner in WT mice and worsened in HD cells and mice, leading to reduced PSD-95 levels. These results demonstrate a direct role of HSF1 in synaptic gene regulation that has important implications in synapse maintenance in basal and pathological conditions.

HSF1 is involved in suppressing A1 phenotype conversion of astrocytes following spinal cord injury in rats

BackgroundTwo activation states of reactive astrocytes termed A1 and A2 subtypes emerge at the lesion sites following spinal cord injury (SCI). A1 astrocytes are known to be neurotoxic that participate in neuropathogenesis, whereas A2 astrocytes have been assigned the neuroprotective activity. Heat shock transcription factor 1 (HSF1) plays roles in protecting cells from stress-induced apoptosis and in controlling inflammatory activation. It is unknown whether HSF1 is involved in suppressing the conversion of A1 astrocytes following SCI.MethodsA contusion model of the rat spinal cord was established, and the correlations between HSF1 expression and onset of A1 and A2 astrocytes were assayed by Western blot and immunohistochemistry. 17-AAG, the agonist of HSF1, was employed to treat the primary cultured astrocytes following a challenge by an A1-astrocyte-conditioned medium (ACM) containing 3 ng/ml of IL-1α, 30 ng/ml of TNF-α, and 400 ng/ml of C1q for induction of the A1 subtype. The effects of 17-AAG on the phenotype conversion of astrocytes, as well as underlying signal pathways, were examined by Western blot or immunohistochemistry.ResultsThe protein levels of HSF1 were significantly increased at 4 days and 7 days following rat SCI, showing colocalization with astrocytes. Meanwhile, C3-positive A1 astrocytes were observed to accumulate at lesion sites with a peak at 1 day and 4 days. Distinctively, the S100A10-positive A2 subtype reached its peak at 4 days and 7 days. Incubation of the primary astrocytes with ACM markedly induced the conversion of the A1 phenotype, whereas an addition of 17-AAG significantly suppressed such inducible effects without conversion of the A2 subtype. Activation of HSF1 remarkably inhibited the activities of MAPKs and NFκB, which was responsible for the regulation of C3 expression. Administration of 17-AAG at the lesion sites of rats was able to reduce the accumulation of A1 astrocytes.ConclusionCollectively, these data reveal a novel mechanism of astrocyte phenotype conversion following SCI, and HSF1 plays key roles in suppressing excessive increase of neurotoxic A1 astrocytes.

Fto Promotes Extramedullary Progression of Multiple Myeloma By Regulation of HSF1 through m6a RNA Methylation

Introduction :Extramedullary myeloma (EMM), defined by the presence of clonal cells outside the bone marrow, has extraordinarily heterogeneous biological and clinical features and is associated with an adverse prognosis. Recent studies have shown the upregulation of heat shock transcription factor 1 (HSF1) in EMM (Heimberger et al, Brit J Haematol 2013), but little is known about its role and mechanisms underlying the regulation. There is growing evidence that dysregulation of N6-methyladenosine (m6A) RNA methylation plays an important role in tumor invasion and metastasis (Pan et al, J Hematol Oncol 2018) and the m6A RNA modifications are involved in the heat shock response. This study was designed to reveal that the fat mass and obesity-associate protein (FTO), as a major m6A demethylase, plays a critical oncogenic role in extramedullary metastasis in multiple myeloma (MM) through regulating HSF1 and heat shock proteins (HSPs). Methods and results: Using transcriptome sequencing, we determined that FTO was significantly upregulated in plasma cells (PCs) from EMM and MM patients, compared to healthy donors. We then evaluated the expression levels of FTO and HSF1 in plasma cells (PCs) from EMM patients by qRT-PCR and western blot analysis. The results verified that FTO and HSF1 were markedly upregulated in PCs from newly diagnosed MM patients compared to healthy donors, and it was further upregulated in PCs from patients with EMM. And the expression levels of FTO was positively correlated with HSF1. Measured by colorimetric assay, we found that m6A RNA methylation level of EMM patients was lower than the level of MM and healthy donors, which was consistent with the expression of FTO in EMM patients. Importantly, enforced expression or knockdown expression of FTO via lentivirus transfection, dramatically influenced the proliferation, migration and invasion in MM cells. m6A RNA methylation has been reported to been associated with heat shock response (Zhou et al, Nature 2015). We therefore examined whether FTO can regulate HSF1 in EMM. Enforced expression of FTO dramatically elevated the level of HSF1 and HSPs in MM.1R and RPMI8226 cells, while inhibition of FTO reduced the level of HSF1 and HSPs. Through m6A sequencing and RNA sequencing, we confirmed that HSF1 was a novel target of FTO. Indeed, the demethylase FTO could positively regulate the expression of HSF1 at both the mRNA and protein levels in MM cells. In addition, knockdown of HSF1 by siRNA markedly reduced the ability of migration and invasion of MM cells overexpressing FTO. Thus, these results suggest that HSF1 is a key target gene of demethylase FTO in EMM. Moreover, pharmacological inhibitors of FTO, MA and MA2, could significantly enhance the effect of bortezomib in MM cells. Recent studies have suggested that for m6A modification to exert its biological functions, it must first be recognized by m6A reader proteins. Here, we evaluated whether demethylase FTO could regulate the level of HSF1 via YTHDF2, a crucial m6A reader protein. We confirmed with colorimetric assay that overexpression of FTO could reduce m6A levels in MM cells, and knockdown of FTO could enhance the m6A levels. Using RNA immuno-precipitation sequence (RIP-seq), we identified direct binding between the 3' end of the HSF1 transcript and the m6A reader protein YTHDF2. Consistently, we showed that silencing of YTHDF2 with siRNA strongly promoted HSF1 expression in MM cells with knockdown of FTO. In addition, by RNA stability analysis, we found that the YTHDF2 could regulate the expression of HSF1 through affecting the stability of HSF1. Next, we evaluated whether FTO could regulate extramedullary metastasis of MM in vivo. NCG mice were injected via tail vein with RPMI-8226-luc cells to establish EMM xenografts. Mice were assessed by in vivo bioluminescent imaging and PET-CT. The results showed that combined treatment with MA2 plus bortezomib could remarkably reduce the extramedullary metastasis in MM. Conclusions: Our datas reveal that FTO is frequently up-regulated in EMM and contributes to MM progression. FTO promotes HSF1 expression through inhibiting the degradation effect of YTHDF2 by reducing m6A levels in these mRNA transcripts. Therefore, FTO-YTHDF2-HSF1 regulatory axis may potentially be applied in EMM treatment. Disclosures No relevant conflicts of interest to declare.

Evaluation of in vitro and in vivo therapeutic antitumor efficacy of transduction of polo-like kinase 1 and heat shock transcription factor 1 small interfering RNA.

Mitotic progression is regulated by the phosphorylation of heat shock transcription factor 1 (HSF1) by polo-like kinase 1 (PLK1); however, this interaction is often deregulated in tumors. High expression levels of PLK1 and HSF1 have been observed in various types of human cancer. In the present study, it was investigated whether small interfering (si)RNA against PLK1 or HSF1 could suppress tumor growth in vitro and in vivo. In vitro transfection of PLK1 and HSF1 siRNA into PKL1- and HSF1-positive human breast tumor MDA-MB-231 and human cervical carcinoma HeLa cells inhibited cell growth via suppression of PLK1 and HSF1 mRNA expression, respectively. However, the transfection of PLK1 or HSF1 siRNA did not significantly affect the cytotoxicity of doxorubicin in HeLa cells. Furthermore, injection of PKL1 or HSF1 siRNA into mice with liver HeLa metastasis suppressed tumor growth. From these findings, PLK1 and HSF1 may be considered to be promising targets for antitumor therapy.

Heat shock transcription factor 1 promotes the proliferation, migration and invasion of osteosarcoma cells.

Osteosarcoma is the most commonly diagnosed primary malignancy of bone and its overall survival rate is still very low. The molecular mechanisms underlying the progression of osteosarcoma have not been clearly illuminated. Heat shock transcription factor 1 (HSF1) is a key regulator of the heat shock response and also plays important roles in many cancers, but its function in osteosarcoma remains unexplored. In this study, the proliferation of osteosarcoma cells was determined by Cell Counting Kit-8 assays and colony formation assays. Transwell assays were used to demonstrate the migration and invasion abilities of osteosarcoma cells. A tumour formation assay in a nude mouse model was performed to assess the effect of HSF1 on osteosarcoma cell growth in vivo. The protein levels of HSF1 were analysed with immunohistochemical staining in samples from osteosarcoma patients. We demonstrated that knockdown of HSF1 reduced the proliferation, migration and invasion of osteosarcoma cells, while overexpression of HSF1 promoted the proliferation, migration and invasion of osteosarcoma cells. Furthermore, HSF1 promoted the proliferation of osteosarcoma cells in vivo. In addition, high levels of HSF1 were associated with a poor prognosis in osteosarcoma. These data highlight an important role of HSF1 in proliferation, migration and invasion of osteosarcoma cells. Moreover, the expression of HSF1 was associated with prognosis in osteosarcoma.

Abnormal degradation of the neuronal stress-protective transcription factor HSF1 in Huntington\u2019s disease

Huntington’s Disease (HD) is a neurodegenerative disease caused by poly-glutamine expansion in the Htt protein, resulting in Htt misfolding and cell death. Expression of the cellular protein folding and pro-survival machinery by heat shock transcription factor 1 (HSF1) ameliorates biochemical and neurobiological defects caused by protein misfolding. We report that HSF1 is degraded in cells and mice expressing mutant Htt, in medium spiny neurons derived from human HD iPSCs and in brain samples from patients with HD. Mutant Htt increases CK2α′ kinase and Fbxw7 E3 ligase levels, phosphorylating HSF1 and promoting its proteasomal degradation. An HD mouse model heterozygous for CK2α′ shows increased HSF1 and chaperone levels, maintenance of striatal excitatory synapses, clearance of Htt aggregates and preserves body mass compared with HD mice homozygous for CK2α′. These results reveal a pathway that could be modulated to prevent neuronal dysfunction and muscle wasting caused by protein misfolding in HD.

Upregulation of HSF1 in estrogen receptor positive breast cancer.

Heat shock transcription factor 1 (HSF1), a key regulator of the heat-shock response, is deregulated in many cancers. HSF1 can mediate cancer cell survival and metastasis. High levels of HSF1 have been associated with poor prognosis in breast cancer. The nature of HSF1 upregulation needs to be validated in different cohorts to further validate its prognostic utility in breast cancer.We first evaluated its expression in a cohort of breast cancer tissue microarrays with Oncotype DX recurrence scores available using immunohistochemistry. To further confirm the clinical relevance and prognostic impact, mutational and methylation status of the gene were also assessed in The Cancer Genome Atlas and publically available microarray datasets.Immunohistochemical analysis showed that HSF1 expression is independent of Oncotype DX high recurrence score in ER-positive node-negative patients. Analysis of The Cancer Genome Atlas data revealed upregulation of HSF1 is not due to methylation or mutation. HSF1 copy number variations and amplifications (15%) were not associated with survival. In publicly available microarray datasets, a prognostic impact was observed in ER-positive tumors, but not in ER-negative tumors. Patients with ER-positive tumors with high HSF1 levels were associated with shorter overall survival (P = 0.00045) and relapse-free survival (P = 0.0057). In multivariable analysis, HSF1 remained a significant prognostic parameter.The mRNA expression levels of HSF1 in ER-positive breast cancer are associated with both shorter relapse-free and overall survival. This prognostic impact is specific to mRNA expression, but stayed insignificant by protein expression or by analyzing amplification events.

Exercise preconditioning attenuates pressure overload-induced pathological cardiac hypertrophy: potential role of HSF1 and NF-κB p65 signaling

To observe the effect of exercise preconditioning (EP) on pressure overload-induced pathological cardiac hypertrophy and explore related mechanisms. Ten-week-old male Sprague-Dawley rats (n = 80) were randomly divided into four groups via random number table method: sham, TAC, EP + sham and EP + TAC. Two EP groups were subjected to 4 weeks of treadmill training, and followed by sham and TAC operations. Eight weeks after the surgery, mean arterial pressure (MAP), cardiac morphology, mRNA expressions of the B-type natriuretic peptide (BNP) and heat shock protein (HSP) 70 and protein expression of the BNP, heat shock transcription factor 1 (HSF1), HSP70, nuclear factor κB (NF-κB) p65, and interleukin-2 (IL-2) were examined. (1) Pathological cardiac hypertrophy index: eight weeks after TAC, MAP, heart size, HW/BW, cross-sectional area of the cardiomyocytes (CSA) and mRNA and protein expressions of BNP in the LV were all significantly higher in the TAC and EP + TAC groups than respective sham groups (all P < 0.05). HW/BW, CSA, and mRNA and protein expressions of BNP in the LV were significantly lower in EP + TAC group than in TAC group (all P < 0.05). (2) mRNA and protein expressions of HSF1 and HSP70 and nuclear HSF1 levels were significantly downregulated post TAC, however, EP treatment significantly increased the expression of HSF1 and nuclear HSF1 levels in TAC rats (all P < 0.05). (3) mRNA and protein expressions of NF-κB p65 and IL-2 were significantly increased in the TAC and EP + TAC groups compared with the respective sham groups (all P < 0.05), which were significantly downregulated in EP + TAC group compared to TAC group (all P < 0.05). EP could effectively reduce the cardiac hypertrophic responses induced by TAC possibly through upregulating the expressions of HSF1 and HSP70 and inhibiting the expression of NF-κB p65 and its nuclear translocation.

Abstract 4731: High level of nuclear heat-shock factor 1 is associated with aggressive disease and suggests targets for therapy in endometrial carcinoma

Abstract Background: The transcriptional regulator heat-shock transcription factor 1 (HSF1) has recently been linked to progression of breast cancer (Santagata, PNAS 2011). HSF1 is a regulator of cellular response to stress and drives the production of heat shock proteins (HSPs). The role of HSF1 to promote tumor cell survival has been demonstrated in hsf1 knockout mice (Dai, Cell 2007). Furthermore, recent identification of a specific role of HSF1 in cancer progression has led to new relevance of HSF1 as a marker both for prognosis and as a predictive marker. The role of HSF1 in endometrial cancer has so far been unexplored. Methods: A total of 823 lesions from endometrial carcinoma precursors, primary tumors and metastases were prospectively collected and explored for HSF1 protein expression in relation to established markers for aggressive disease and survival. Transcriptional alterations related to HSF1 protein level were investigated by microarray analysis for 224 freshly frozen samples in parallel. Results: High expression of HSF1 protein in endometrial carcinoma is significantly associated with non-endometrioid histology, high grade, ERα loss and poor survival (all p-values ≤0.02). Also among ERα-positive patients presumed to have good prognosis, high expression of HSF1 is correlated with poor survival (p=0.02). HSF1 mRNA and protein levels increase significantly from primary tumors to metastases (p&amp;lt;0.03). HSF1-related gene signatures were explored. Such signatures increase from complex atypical hyperplasias through primary tumors to metastatic lesion and were also found to have prognostic value. HSF1 immunohistochemistry suggests the use of HSP90 inhibitiors as potential novel therapeutic approach in cases with high protein expression of HSF1. Conclusion: We demonstrate for the first time in endometrial cancer that high expression of HSF1 and measures for transcriptional activation of HSF1 associates with poor outcome, progression from precursor lesions through primary tumors to metastatic lesions. Also, HSP90 inhibitors are suggested as potential new targeted therapeutics for cases with high HSF1 levels in particular. 1. Santagata,S., et al., High levels of nuclear heat-shock factor 1 (HSF1) are associated with poor prognosis in breast cancer. Proc Natl Acad Sci U S A, 2011. 108(45):p. 18378-83. 2. Dai,C., et al., Heat shock factor 1 is a powerful multifaceted modifier of carcinogenesis. Cell, 2007. 130(6): p. 1005-18. Citation Format: Hilde Engerud, Ingvild Løberg Tangen, Anna Berg, Kanthida Kusonmano, Ingunn Stefansson, Helga B. Salvesen, Camilla Krakstad. High level of nuclear heat-shock factor 1 is associated with aggressive disease and suggests targets for therapy in endometrial carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4731. doi:10.1158/1538-7445.AM2014-4731

Changes in cardiac thrombomodulin and heat shock transcription factor 1 expression and peripheral thrombomodulin and catecholamines during hypothermia in rats

Effects of hypothermia and rewarming on thrombomodulin, catecholamines and heat shock transcription factor 1 (HSF1) were studied in rats. The aims of this study were to clarify whether cold stress, under anesthesia, is sufficient to change levels of thrombomodulin in healthy endothelium and in the circulation and whether adrenaline, noradrenaline and HSF1 could act as regulators in the process. Rats were divided into control, mild hypothermia (2 and 4.5 hours at + 21 °C; MH1, MH2), severe hypothermia (2 and 4.5 h at + 10 °C; SH1, SH2) and two rewarming groups (2 h at + 10 °C followed by 2 h at + 21 °C or 3 h at + 28 °C; SHW1, SHW2) (n = 15/group, except n = 6 in MH1). Fentanyl-fluanisone-midazolam was used as anesthetic. Low levels of thrombomodulin in plasma and myocardial arterioles/venules measured by ELISA and immunohistochemistry were associated with significant increase of thrombomodulin transcript level in SH1 rats analyzed by quantitative PCR. Plasma adrenaline correlated negatively with the relative amount of myocardial thrombomodulin transcripts and positively with plasma thrombomodulin in SH. Transcript levels of thrombomodulin and HSF1 correlated strongly (r = 0.83; p < 0.001) in SH. Plasma/urine ratio of thrombomodulin and plasma adrenaline (r = 0.87; p = 0.005) or noradrenaline (r = 0.78; p = 0.023) were strongly correlated in SHW1 rats. Hence, cellular and soluble levels of thrombomodulin are modified by cold stress in healthy rats, possibly via catecholamines and HSF1.

Overexpression of heat shock transcription factor 1 enhances the resistance of melanoma cells to doxorubicin and paclitaxel

BackgroundHeat Shock Transcription Factor 1 (HSF1) is activated under stress conditions. In turn, it induces expression of Heat Shock Proteins (HSPs), which are well-known regulators of protein homeostasis. Elevated levels of HSF1 and HSPs were observed in many types of tumors. The aim of the present study was to determine whether HSF1 could have an effect on the survival of cancer cells treated with chemotherapeutic cytotoxic agents.MethodsWe constructed mouse (B16F10) and human (1205Lu, WM793B) melanoma cells overexpressing full or mutant form of human HSF1: a constitutively active one with a deletion in regulatory domain or a dominant negative one with a deletion in the activation domain. The impact of different forms of HSF1 on the expression of HSP and ABC genes was studied by RT-PCR and Western blotting. Cell cultures were treated with increasing amounts of doxorubicin, paclitaxel, cisplatin, vinblastine or bortezomib. Cell viability was determined by MTT, and IC50 was calculated. Cellular accumulation of fluorescent dyes and side population cells were studied using flow cytometry.ResultsCells overexpressing HSF1 and characterized by increased HSPs accumulation were more resistant to doxorubicin or paclitaxel, but not to cisplatin, vinblastine or bortezomib. This resistance correlated with the enhanced efflux of fluorescent dyes and the increased number of side population cells. The expression of constitutively active mutant HSF1, also resulting in HSPs overproduction, did not reduce the sensitivity of melanoma cells to drugs, unlike in the case of dominant negative form expression. Cells overexpressing a full or dominant negative form of HSF1, but not a constitutively active one, had higher transcription levels of ABC genes when compared to control cells.ConclusionsHSF1 overexpression facilitates the survival of melanoma cells treated with doxorubicin or paclitaxel. However, HSF1-mediated chemoresistance is not dependent on HSPs accumulation but on an increased potential for drug efflux by ABC transporters. Direct transcriptional activity of HSF1 is not necessary for increased expression of ABC genes, which is probably mediated by HSF1 regulatory domain.

Impaired Heat Shock Response in Cells Expressing Full-Length Polyglutamine-Expanded Huntingtin

The molecular mechanisms by which polyglutamine (polyQ)-expanded huntingtin (Htt) causes neurodegeneration in Huntington's disease (HD) remain unclear. The malfunction of cellular proteostasis has been suggested as central in HD pathogenesis and also as a target of therapeutic interventions for the treatment of HD. We present results that offer a previously unexplored perspective regarding impaired proteostasis in HD. We find that, under non-stress conditions, the proteostatic capacity of cells expressing full length polyQ-expanded Htt is adequate. Yet, under stress conditions, the presence of polyQ-expanded Htt impairs the heat shock response, a key component of cellular proteostasis. This impaired heat shock response results in a reduced capacity to withstand the damage caused by cellular stress. We demonstrate that in cells expressing polyQ-expanded Htt the levels of heat shock transcription factor 1 (HSF1) are reduced, and, as a consequence, these cells have an impaired a heat shock response. Also, we found reduced HSF1 and HSP70 levels in the striata of HD knock-in mice when compared to wild-type mice. Our results suggests that full length, non-aggregated polyQ-expanded Htt blocks the effective induction of the heat shock response under stress conditions and may thus trigger the accumulation of cellular damage during the course of HD pathogenesis.

Hyperthermia‐associated carboplatin resistance: Differential role of p53, HSF1 and Hsp70 in hepatoma cells

Due to substantial technical improvements, clinical application of heat as a co-adjuvant in cancer treatment is acquiring new interest. The effect of hyperthermia on hepatoma cell lines Hep3B (p53 defective) and HepG2 (p53 wild type) when investigated led to an interesting observation that Hep3B cells are more susceptible to heat stress than HepG2 cells. In addition, heat-induced carboplatin resistance was observed in HepG2 cells only. To investigate the reasons, heat shock response was explored and it was observed that heat stress augmented heat shock protein 70 (Hsp70) expression levels in HepG2 and not in Hep3B cells. Furthermore, in HepG2 cells, induced Hsp70 is regulated by both p53 and heat shock transcription factor 1 (HSF1) wherein HSF1 levels are modulated by p53. The data implies that Hep3B are more susceptible to death upon heat stress than HepG2 cells because of non-induction of Hsp70. In addition, it was observed that inhibition of heat-induced p53/HSF1 diminishes Hsp70 levels, thereby restoring the sensitivity of heat-stressed HepG2 cells to carboplatin-triggered cell death. Collectively, the present study establishes interplay of p53, HSF1, and Hsp70 upon heat stress in HepG2 cells and also defines novel strategies to overcome constraints of utility of hyperthermia in cancer therapy through p53/HSF1-targeted therapeutic intervention.

Heat shock protein expression is low in intestinal mucosa in juvenile idiopathic arthritis: a defect in immunoregulation?

Objectives: Heat shock proteins (HSPs) are involved in the regulation of inflammation and in the maintenance of mucosal integrity. Their altered expression may be a marker of mucosal inflammation and also contribute to tissue injury. The small intestinal mucosa in children with juvenile idiopathic arthritis (JIA) shows signs of intestinal immune activation, such as increased intraepithelial cytotoxic lymphocyte counts. To further evaluate the characteristics of this immune activation in JIA, we have studied the expression of several HSPs, major histocompatibility complex (MHC) class I-related chain A (MICA), and the heat shock transcription factor 1 (HSF1) in intestinal biopsies from children with JIA.Methods: We studied 15 patients with JIA. Controls included 13 children without JIA, studied for various gastrointestinal (GI) symptoms, but eventually shown not to have any GI disease. The subjects were examined by endoscopy. The expression of HSP60, HSP70, MICA, and HSF1 was analysed in ileal and duodenal biopsies by using immunohistochemistry.Results: The expression levels of HSP60, MICA, and HSF1 were significantly lower in the duodenal epithelium in the JIA patients compared to the controls. MICA and HSF1 also showed lower expression in the ileal epithelium. The expression of HSP70 did not differ between the groups.Conclusions: The downregulation of HSP60, MICA, and HSF1 in small intestinal mucosa may indicate that intestinal epithelial cells show immune aberration in JIA. We speculate that the low heat shock response may play a role in the pathogenesis of JIA, interfering with mucosal integrity and local intestinal immunoregulation.

Glycogen synthase kinase-3β activity plays very important roles in determining the fate of oxidative stress-inflicted neuronal cells

Glycogen synthase kinase-3, especially the beta form (GSK-3β), plays key roles in oxidative stress-induced neuronal cell death, an important pathogenic mechanism of various neurodegenerative diseases. Although the neuroprotective effects of GSK-3β inhibitors have been described, the optimal level of GSK-3β inhibition for neuronal cell survival has not yet been determined. We investigated the effect of varying GSK-3β activity on the viability of oxidative stress-injured neuronally differentiated PC12 (nPC12) cells and intracellular signals related with the GSK-3β and caspase-3 pathways. Compared to the nPC12 control cells treated with only 100 μM H 2O 2, treatment of 50–200 nM GSK-3β inhibitor II or 25–500 nM GSK-3β inhibitor VIII reduced the increased enzyme activity by about 50% and protected the cells against H 2O 2-induced oxidative stress. The optimal concentration of GSK-3β inhibitor II enhanced heat shock transcription factor-1 levels, decreased levels of phosphorylated tau (Ser202) and cytosolic cytochrome c, activated caspase-3, and cleaved poly (ADP-ribose) polymerase. In contrast, higher concentrations of GSK-3β inhibitor II (more than 500 nM) induced neuronal cell death and showed opposite effects relative to the above described intracellular signals. These results suggest that optimized inhibitor levels for modulating GSK-3β activity may prevent apoptosis induced by oxidative stress associated with neurodegenerative diseases.

HSF1 Down-regulates XAF1 through Transcriptional Regulation

Studies have indicated the role of HSF1 (heat-shock transcription factor 1) in repressing the transcription of some nonheat shock genes. XAF1 (XIAP-associated factor 1) was an inhibitor of apoptosis-interacting protein with the effect of antagonizing the cytoprotective role of XIAP. XAF1 expression was lower in gastrointestinal cancers than in normal tissues with the mechanism unclear. Here we showed that gastrointestinal cancer tissues expressed higher levels of HSF1 than matched normal tissues. The expression of XAF1 and HSF1 was negatively correlated in gastrointestinal cancer cell lines. Stress stimuli, including heat, hypo-osmolarity, and H2O2, significantly suppressed the expression of XAF1, whereas the alteration of HSF1 expression negatively correlated with XAF1 expression. We cloned varying lengths of the 5'-flanking region of the XAF1 gene into luciferase reporter vectors, and we evaluated their promoter activities. A transcription silencer was found between the -592- and -1414-nucleotide region that was rich in nGAAn/nT-TCn elements (where n indicates G, A, T, or C). A high affinity and functional HSF1-binding element within the -862/-821-nucleotide region was determined by electrophoretic mobility shift assay and chromatin immunoprecipitation assay. Inactivation of this "heat-shock element" by either site-directed mutation or an HSF1 inhibitor, pifithrin-alpha, restored the promoter activity of the silencer structure. Moreover, pretreatment with antioxidants suppressed HSF1 binding activity and increased the transcriptional activity and expression of XAF1. These findings suggested that endogenous stress pressure in cancer cells sustained the high level expression of HSF1 and subsequently suppressed XAF1 expression, implicating the synergized effect of two anti-apoptotic protein families, HSP and inhibitors of apoptosis, in cytoprotection under stress circumstances.

Interspecific- and acclimation-induced variation in levels of heat-shock proteins 70 (hsp70) and 90 (hsp90) and heat-shock transcription factor-1 (HSF1) in congeneric marine snails (genus Tegula): implications for regulation of hsp gene expression.

In our previous studies of heat-shock protein (hsp) expression in congeneric marine gastropods of the genus Tegula, we observed interspecific and acclimation-induced variation in the temperatures at which heat-shock gene expression is induced (T(on)). To investigate the factors responsible for these inter- and intraspecific differences in T(on), we tested the predictions of the 'cellular thermometer' model for the transcriptional regulation of hsp expression. According to this model, hsps not active in chaperoning unfolded proteins bind to a transcription factor, heat-shock factor-1 (HSF1), thereby reducing the levels of free HSF1 that are available to bind to the heat-shock element, a regulatory element upstream of hsp genes. Under stress, hsps bind to denatured proteins, releasing HSF1, which can now activate hsp gene transcription. Thus, elevated levels of heat-shock proteins of the 40, 70 and 90 kDa families (hsp 40, hsp70 and hsp90, respectively) would be predicted to elevate T(on). Conversely, elevated levels of HSF1 would be predicted to decrease T(on). Following laboratory acclimation to 13, 18 and 23 degrees C, we used solid-phase immunochemistry (western analysis) to quantify endogenous levels of two hsp70 isoforms (hsp74 and hsp72), hsp90 and HSF1 in the low- to mid-intertidal species Tegula funebralis and in two subtidal to low-intertidal congeners, T. brunnea and T. montereyi. We found higher endogenous levels of hsp72 (a strongly heat-induced isoform) at 13 and 18 degrees C in T. funebralis in comparison with T. brunnea and T. montereyi. However, T. funebralis also had higher levels of HSF1 than its congeners. The higher levels of HSF1 in T. funebralis cannot, within the framework of the cellular thermometer model, account for the higher T(on) observed for this species, although they may explain why T. funebralis is able to induce the heat-shock response more rapidly than T. brunnea. However, the cellular thermometer model does appear to explain the cause of the increases in T(on) that occurred during warm acclimation of the two subtidal species, in which warm acclimation was accompanied by increased levels of hsp72, hsp74 and hsp90, whereas levels of HSF1 remained stable. T. funebralis, which experiences greater heat stress than its subtidal congeners, consistently had higher ratios of hsp72 to hsp74 than its congeners, although the sum of levels of the two isoforms was similar for all three species except at the highest acclimation temperature (23 degrees C). The ratio of hsp72 to hsp74 may provide a more accurate estimate of environmental heat stress than the total concentrations of both hsp70 isoforms.

Age-Related Alterations in the Activation of Heat Shock Transcription Factor 1 in Rat Hepatocytes

The induction of hsp70 transcription by heat shock is significantly reduced in hepatocytes isolated from old rats compared to hepatocytes isolated from young/adult rats, and the decline in hsp70 transcription is correlated with a decrease in the induction of heat shock transcription factor 1 (HSF1) binding to the heat shock element. However, the decreased HSF1 binding activity to DNA is not due to reduced levels of HSF1 that are available for activation by heat shock. In fact, the levels of HSF1 are two- to threefold higher in hepatocytes from old rats, and the age-related increase in the levels of HSF1 protein in hepatocytes appears to arise from a decrease in the degradation of the HSF1 because HSF1 mRNA levels do not change and the synthesis of HSF1 decreases approximately 50% with age. No evidence was found for an impairment in HSF1 oligomerization in hepatocytes from old rats, e.g., the level of HSF1 trimers, the nuclear translocation of HSF1, and the phosphorylation of HSF1 after heat shock are similar in hepatocytes isolated from young/adult and old rats. However, the thermostability of the DNA binding activity of HSF1 was significantly reduced with age in a cell-free system as well as in isolated hepatocytes.