Role Of Heat Shock Factor 1 Research Articles

Heat shock factor 2 is a stress-responsive mediator of neuronal migration defects in models of fetal alcohol syndrome.

Fetal alcohol spectrum disorder (FASD) is a frequent cause of mental retardation. However, the molecular mechanisms underlying brain development defects induced by maternal alcohol consumption during pregnancy are unclear. We used normal and Hsf2-deficient mice and cell systems to uncover a pivotal role for heat shock factor 2 (HSF2) in radial neuronal migration defects in the cortex, a hallmark of fetal alcohol exposure. Upon fetal alcohol exposure, HSF2 is essential for the triggering of HSF1 activation, which is accompanied by distinctive post-translational modifications, and HSF2 steers the formation of atypical alcohol-specific HSF1–HSF2 heterocomplexes. This perturbs the in vivo binding of HSF2 to heat shock elements (HSEs) in genes that control neuronal migration in normal conditions, such as p35 or the MAPs (microtubule-associated proteins, such as Dclk1 and Dcx), and alters their expression. In the absence of HSF2, migration defects as well as alterations in gene expression are reduced. Thus, HSF2, as a sensor for alcohol stress in the fetal brain, acts as a mediator of the neuronal migration defects associated with FASD.Subject Categories Development & Differentiation; Neuroscience

Roles of heat shock factor 1 in neuronal response to fetal environmental risks and its relevance to brain disorders.

Prenatal exposure of the developing brain to various environmental challenges increases susceptibility to late onset of neuropsychiatric dysfunction; still, the underlying mechanisms remain obscure. Here we show that exposure of embryos to a variety of environmental factors such as alcohol, methylmercury, and maternal seizure activates HSF1 in cerebral cortical cells. Furthermore, Hsf1 deficiency in the mouse cortex exposed in utero to subthreshold levels of these challenges causes structural abnormalities and increases seizure susceptibility after birth. In addition, we found that human neural progenitor cells differentiated from induced pluripotent stem cells derived from schizophrenia patients show higher variability in the levels of HSF1 activation induced by environmental challenges compared to controls. We propose that HSF1 plays a crucial role in the response of brain cells to prenatal environmental insults and may be a key component in the pathogenesis of late-onset neuropsychiatric disorders.

Pattern of heat shock factor and heat shock protein expression in lymphocytes of bipolar patients: Increased HSP70-glucocorticoid receptor heterocomplex

Bipolar disorder (BD), a stress-related disease, is characterized by altered glucocorticoid receptor (GR) signalling. Stress response includes activation of heat shock factor (HSF) and subsequent heat shock protein (HSP) synthesis which regulate GR folding and function. The objective of this study was to investigate the possible role of HSFs, HSPs and their interaction with GR in BD. We applied immunoprecipitation, SDS-PAGE/Western blot analysis and electrophoretic mobility shift assay (EMSA) in lymphocytes (whole cell or nuclear extracts) from BD patients and healthy subjects and determined the HSPs (HSP90 and HSP70), the heterocomplexes HSP90-GR and HSP70-GR, the HSFs (HSF1 and HSF4) as well as the HSF-DNA binding. The HSP70-GR heterocomplex was elevated (p < 0.05) in BD patients vs healthy subjects, and nuclear HSP70 was reduced (p ≤ 0.01) in bipolar manic patients. Protein levels of HSF1, HSF4, HSP90, HSP90-GR heterocomplex, and HSF-DNA binding remained unaltered in BD patients vs healthy subjects. The corresponding effect sizes (ES) indicated a large ES for HSP70-GR, HSP70, HSF-DNA binding and HSF4, and a medium ES for HSP90, HSF1 and HSP90-GR between healthy subjects and bipolar patients. Significant correlations among HSFs, HSPs, GR and HSP70-GR heterocomplex were observed in healthy subjects, which were abrogated in bipolar patients. The higher interaction between GR and HSP70 and the disturbances in the relations among heat shock response parameters and GR as observed in our BD patients may provide novel insights into the contribution of these factors in BD aetiopathogenesis.

Abstract 2933: HSP90 inhibiting anti-cancer therapeutics enhance bone loss by increasing osteoclast formation: Mechanism of action.

Abstract Breast cancer cells metastasize to bone where they cause bone destruction. Here, cancer cells promote bone loss through the recruitment of osteoclasts, specialised macrophage-like cells that resorb bone. Bone resorption releases bone matrix factors that increase tumor growth, resulting in vicious cycle of bone resorption and tumor progression. Previously we found 17-AAG, an Hsp90 inhibitor and anti-cancer therapeutic significantly increases MDA-MB-231 breast cancer cell growth in bone following intracardiac inoculation in nude mice. 17-AAG also increases osteoclast formation in vitro and in vivo even though Hsp90 client proteins are important in osteoclast formation. In addition to degradation of Hsp90 client proteins, inhibition by 17-AAG also causes cell stress responses through the activation of Heat Shock Factor 1 (HSF1). Therefore we sought to characterize 17-AAG and new HSP90 inhibitors, to examine the role of HSF1 in their effects and to determine the molecular mechanism of 17-AAG. 17-AAG and new, but structurally unrelated, Hsp90 blockers CCT018159 and NVP-AUY922 dose-dependently enhanced osteoclast formation from mouse bone marrow and RAW264.7 cells. Ablating HSF1 action by shRNA transfection and by pharmacological inhibition using KNK437 inhibited 17-AAG effects upon osteoclast formation. Osteoclast formation from HSF1 knockout mouse bone marrow were unresponsive to 17-AAG, but wild type cells did respond. To determine the mechanism of how 17-AAG is increasing osteoclast formation, the effect of 17-AAG upon critical transcription factors in the osteoclast formation pathway was observed. 17-AAG treatment did not affect transcriptional activity of NFκB, AP-1 or NFATc1. Moreover 17-AAG did not increase NFATc1 or cFos protein expression over time. In contrast, 17-AAG treatment increased protein expression of MITF,in a dose and time dependent manner. RAW264.7 cells were then transiently transfected with a luciferase reporter construct containing the promoter region of a MITF target gene v-ATPase-d2. 17-AAG treatment increased the transcriptional activity of this promoter. RANKL and 17-AAG combination treatment increased reporter activity of the v-ATPase-d2 promoter in an additive manner. The stress induced kinase p38 is known to be important in osteoclast formation. Our results show inhibiting p38 with SB203580 decreases MITF protein expression. These data indicate that Hsp90 inhibition causes an increase in osteoclast formation in an HSF1-dependent manner. The data provides a mechanism of action, whereby 17-AAG is increasing osteoclast numbers, through the increased protein expression of Mitf. Citation Format: A. Gabrielle J. van der Kraan, Ryan R. Chai, Michelle M. Kouspou, Ben J. Lang, Preetinder P. Singh, Jiake Xu, Damien Eeles, Matthew T. Gillespie, Julian M. Quinn, John T. Price. HSP90 inhibiting anti-cancer therapeutics enhance bone loss by increasing osteoclast formation: Mechanism of action. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2933. doi:10.1158/1538-7445.AM2013-2933

The effect of HSF‐1 and HSP70 on autophagy regulation

Autophagy is an evolutionarily conserved mechanism that plays an important role in maintaining cellular homeostasis by recycling large protein aggregates, damaged proteins, entire organelles, and bulk of cytoplasm within double membrane vacuoles delivered for degradation to lysosomes. Heat stress response renders protection to macromolecules, cells, as well as whole organism by assisting in folding of newly synthesized proteins, in stabilizing of unfolded protein, and in degradation and reactivation of damaged proteins. Previously, we have reported that autophagy is regulated by heat stress response. The purpose of the study was to investigate the role of heat shock factor 1 (HSF‐1), the central regulator of heat shock response and heat shock protein 70 (HSP70), principal effector of heat shock response on starvation‐induced autophagy in A549 cells. We utilized HSF‐1 siRNA to inhibit HSF‐1 protein expression and adenovirus to overexpress inducible form of HSP70. Their effects on starvation‐ induced autophagy were tested. Exposure to HSF‐1 siRNA augmented the starvation‐induced increase in autophagy and HSP70 significantly blocked this increase. We conclude that heat stress response exert a regulatory role on autophagy.

Abstract LB-488: Induction of autophagy by heat shock factor 1 promotes breast cancer resistance to chemotherapy

Abstract Breast cancer is the most common type of cancer as well as one of the leading causes of cancer-related deaths in women. The increasing emergence of drug resistance to commonly used chemotherapeutic agents has warranted the development of novel and robust treatment options for cancer patients. Carboplatin, a DNA damaging agent, is one of the therapeutic agents which has shown promising results in breast cancer clinical trials. Carboplatin is a second generation platinum drug, increasingly used in clinic to treat cancer patients because of its reduced toxicity and side effects when compared to Cisplatin. Heat Shock Factor 1 (HSF1) is the master regulator of heat shock responses which has also been shown to regulate certain non-heat shock reponse genes. As a transcription factor HSF1 can function as both an activator and repressor of its target genes, resulting in regulation of critical cellular processes invovled in managing cellular stress. The focus of this study is to identify the role of HSF1 in Carboplatin mediated-sensitivity in breast cancer cells, to provide novel insights to improve treatment strategies for cancer patients. Carboplatin treatment in multiple breast cancer cell lines induced growth inhibition which was significantly enhanced upon HSF1 knockdown. Interestingly, the treatment also induced autophagy in these cells. Autophagy is a type II programmed cell death, which is induced in response to metabolic stress and has been demonstrated to mediate drug resistance, since it can play a protective role and allow cells to survive under stressful conditions. Knockdown of HSF1 along with inhibition of autophagy by 3-Methyladenine (3-MA) led to increase in apoptosis in these cells. Furthermore, HSF1 was required for the induction of autophagy in Carboplatin treated cells. Knockdown of HSF1 by siRNA (transient) and shRNA (stable), inhibited induction of autophagy in the presence of Carboplatin, as measured by the punctate EGFP-LC3 structures as well as acridine orange staining. Chromatin immunoprecipitation (ChIP) assay along with Real time- PCR analysis revealed that HSF1 can regulate autophagy by directly regulating the expression of autophagy related genes in the presence of Carboplatin. These findings indicate that HSF1 activates autophagy in response to the Carboplatin induced cellular stress thus leading to cell survival and reducing sensitivity to Carboplatin. Our data strongly support a critical role for HSF1 in promoting chemoresistance through induction of autophgy. This finding has important implications in the development of targeted therapeutics for overcoming Carboplatin resistance in breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-488. doi:1538-7445.AM2012-LB-488

The role of heat shock factor 1 in transcriptional regulation of high mobility group box

Background: Previous study suggests that high mobility group box 1 (HMGB1) can be a potential late inflammatory mediator. However, whether heat shock factor 1 (HSF1) regulate HMGB1 expression via binding to heat shock element (HSE) is not known. Objective: We investigated the role of HSF1 in the transcriptional regulation of HMGB1 protein. Methods: A probe that included HMGB1 promoter region containing HSE was synthesized for electrophoretic mobility shift assay (EMSA) to determine the binding of HSF1 and HSE in the promoter region of HMGB1 gene. Mutant mouse HMGB1 promoter was prepared by PCR amplification on a template of wild-type plasmid DNA with site-directed mutant primers. The mutant DNA fragments were also inserted into a corresponding plasmid. In addition, luciferase reporter plasmids of HMGB1 promoter were constructed to transfect RAW264.7 cells. After that, luciferase activity was measured to assay the effects of the HSF1 transfection on the promoter activity. Results: EMSA result showed a retardation strap after the coculture of biotin labeled HSF1 binding fragment and nuclear protein extracts. The retardation phenomenon could be competed by unlabeled probe and not by unlabeled mutant probe. A super retardation strap was present after adding HSF1 monoclonal antibody. After the HSE core sites was mutated, the relative luciferase activity of the mutant plasmid decreased by 4.26 folds compared with that in the wild-type (23.54±1.68 vs.100.25±3.26, p Conclusion: HSF1 can bind to the promoter region HSE (-668bp~-651bp) of HMGB1, which down-regulates the expression of HMGB1. Keywords: Heat shock transcription factor 1, high mobility group box 1, mouse, RAW264.7, transcription

The role of heat shock factor 1 in transcriptional regulation of high mobility group box

Background: Previous study suggests that high mobility group box 1 (HMGB1) can be a potential late inflammatory mediator. However, whether heat shock factor 1 (HSF1) regulate HMGB1 expression via binding to heat shock element (HSE) is not known. Objective: We investigated the role of HSF1 in the transcriptional regulation of HMGB1 protein. Methods: A probe that included HMGB1 promoter region containing HSE was synthesized for electrophoretic mobility shift assay (EMSA) to determine the binding of HSF1 and HSE in the promoter region of HMGB1 gene. Mutant mouse HMGB1 promoter was prepared by PCR amplification on a template of wild-type plasmid DNA with site-directed mutant primers. The mutant DNA fragments were also inserted into a corresponding plasmid. In addition, luciferase reporter plasmids of HMGB1 promoter were constructed to transfect RAW264.7 cells. After that, luciferase activity was measured to assay the effects of the HSF1 transfection on the promoter activity. Results: EMSA result showed a retardation strap after the coculture of biotin labeled HSF1 binding fragment and nuclear protein extracts. The retardation phenomenon could be competed by unlabeled probe and not by unlabeled mutant probe. A super retardation strap was present after adding HSF1 monoclonal antibody. After the HSE core sites was mutated, the relative luciferase activity of the mutant plasmid decreased by 4.26 folds compared with that in the wild-type (23.54±1.68 vs.100.25±3.26, p Conclusion: HSF1 can bind to the promoter region HSE (-668bp~-651bp) of HMGB1, which down-regulates the expression of HMGB1. Keywords: Heat shock transcription factor 1, high mobility group box 1, mouse, RAW264.7, transcription

HSF1 Is Essential for the Resistance of Zebrafish Eye and Brain Tissues to Hypoxia/Reperfusion Injury

Ischemia and subsequent reperfusion (IR) produces injury to brain, eye and other tissues, contributing to the progression of important clinical pathologies. The response of cells to IR involves activation of several signaling pathways including those activating hypoxia and heat shock responsive transcription factors. However, specific roles of these responses in limiting cell damage and preventing cell death after IR have not been fully elucidated. Here, we have examined the role of heat shock factor 1 (HSF1) in the response of zebrafish embryos to hypoxia and subsequent return to normoxic conditions (HR) as a model for IR. Heat shock preconditioning elevated heat shock protein expression and protected zebrafish embryo eye and brain tissues against HR-induced apoptosis. These effects were inhibited by translational suppression of HSF1 expression. Reduced expression of HSF1 also increased cell death in brain and eye tissues of embryos subjected to hypoxia and reperfusion without prior heat shock. Surprisingly, reduced expression of HSF1 had only a modest effect on hypoxia-induced expression of Hsp70 and no effect on hypoxia-induced expression of Hsp27. These results establish the zebrafish embryo as a model for the study of ischemic injury in the brain and eye and reveal a critical role for HSF1 in the response of these tissues to HR. Our results also uncouple the role of HSF1 expression from that of Hsp27, a well characterized heat shock protein considered essential for cell survival after hypoxia. Alternative roles for HSF1 are considered.

Abstract 2076: HSF1 inhibition accentuates the lethal activity of proteasome inhibitor in human pancreatic cancer cells

Abstract Presence of aneuploidy increases the intracellular levels of misfolded proteins and proteotoxic stress phenotype in cancer cells, which is exploited and further accentuated by the therapeutic use of a proteasome inhibitor, e.g., the novel, orally bioavailable carfilzomib (CF). This results in the disruption of the cytosolic complex of the histone deacetylase 6 (HDAC6), the ATPase and segregase p97 and the heat shock factor 1 (HSF1)-a transcription factor involved in the transactivation of heat shock proteins (hsps). In the present studies, we demonstrate that treatment of the pancreatic cancer Hs766T and Panc1 cells with CF increased the levels of misfolded polyubiquitylated proteins, which disrupts the repressive HSF1/hsp90/HDAC6/Vp97 complex. This induces the phosphorylation, nuclear localization and activation of HSF1, which transactivated hsps (e.g., hsp70, hsp40 and hsp27) as an adaptive response to CF-induced proteotoxic stress. Treatment with CF also induced endoplasmic reticulum (ER) stress, represented by upregulation of GRP78 and the pro-apoptotic transcription factor CHOP, which induced the BH3 only pro-apoptotic protein Bim. Treatment with CF also dose-dependently decreased the viability of Hs766T and Panc1 cells. To further elucidate the role of HSF1 in the adaptive, protective response to CF-induced proteotoxic stress, we stably knocked down HSF1 in Hs766T cells. As compared to the control cells, following heat shock, Hs766T cells with knockdown (KD) of HSF1 displayed reduced expression of hsps. In the HSF1-KD versus the control Hs766T cells, treatment with CF resulted in a dose-dependent induction of polyubiquitylated proteins, GRP78, CHOP, and Bim, as well as induced significantly more apoptosis. HSF1-KD versus the control Hs766T cells exhibited markedly lower levels of HDAC6 and, consequently, displayed reduced CF-induced perinuclear protective aggresome formation, as determined by confocal microscopy. Additionally, as compared to the control cells, Hs766T cells with HSF1-KD demonstrated higher levels of p-AMPK and CF (5 to 20 nM) or the pan-HDAC inhibitor panobinostat (PS, 50 nM)-induced autophagy, as demonstrated by the accumulation of LC3-II (by immunoblot analysis) and LC3 puncta by immunofluorescent staining. Importantly, co-treatment with the autophagy inhibitor hydroxy-chloroquine (HCQ) induced more CF-induced cell death of HSF1-KD versus the control Hs766T cells. Using an acid-sensitive mCherry-GFP-LC3 reporter, we determined that the induction of LC3-II in the Hs766T HSF1-KD cells is the result of enhanced autophagic flux and not due to inefficient fusion of autophagosomes with lysosomes. Collectively, these findings indicate that inhibition of the transcriptional activity of HSF1 sensitizes pancreatic cancer cells to CF or PS-induced cells death, which is augmented by co-treatment with an autophagy inhibitor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2076. doi:10.1158/1538-7445.AM2011-2076

Prosurvival role of heat shock factor 1 in the pathogenesis of pancreatobiliary tumors.

Several mechanisms have evolved to ensure the survival of cells under adverse conditions. The heat shock response is one such evolutionarily conserved survival mechanism. Heat shock factor-1 (HSF1) is a transcriptional regulator of the heat shock response. By the very nature of its prosurvival function, HSF1 may contribute to the pathogenesis of cancer. The current study investigates the role of HSF1 in the pathogenesis of pancreatobiliary tumors. HSF1 was downregulated in pancreatic cancer (MIA PaCa-2 and S2-013) and cholangiocarcinoma (KMBC and KMCH) cell lines by HSF1-specific small interfering RNA (siRNA). Nonsilencing siRNA was used as control. The effect of HSF1 downregulation on viability and apoptosis parameters, i.e., annexin V, terminal deoxynucleotidyl transferase dUTP-mediated nick end labeling (TUNEL), and caspase-3, was measured. To evaluate the cancer-specific effects of HSF1, the effect of HSF1 downregulation on normal human pancreatic ductal cells was also evaluated. HSF1 is abundantly expressed in human pancreatobiliary cancer cell lines, as well as in pancreatic cancer tissue, as demonstrated by Western blot and immunohistochemistry, respectively. Inhibition of HSF1 expression by the HSF1 siRNA sequences leads to time-dependent death in pancreatic and cholangiocarcinoma cell lines. Downregulation of HSF1 expression induces annexin V and TUNEL positivity and caspase-3 activation, suggesting activation of a caspase-dependent apoptotic pathway. Although caspase-3 inhibition protects against cell death induced by HSF1 expression, it does not completely prevent it, suggesting a role for caspase-independent cell death. HSF1 plays a prosurvival role in the pathogenesis of pancreatobiliary tumors. Modulation of HSF1 activity could therefore emerge as a novel therapeutic strategy for cancer treatment.

Impaired hippocampal spinogenesis and neurogenesis and altered affective behavior in mice lacking heat shock factor 1

Aberrant transcriptional regulation in the brain is thought to be one of the key components of the pathogenesis and pathophysiology of neuropsychiatric disorders. Heat shock factors (HSFs) modulate cellular homeostasis through the control of gene expression. However, the roles of HSFs in brain function have yet to be elucidated fully. In the present study, we attempted to clarify the role of HSF1-mediated gene regulation in neuronal and behavioral development using HSF1-deficient (HSF1(-/-)) mice. We found granule neurons of aberrant morphology and impaired neurogenesis in the dentate gyrus of HSF1(-/-) mice. In addition, HSF1(-/-) mice showed aberrant affective behavior, including reduced anxiety and sociability but increased depression-like behavior and aggression. Furthermore, HSF1 deficiency enhanced behavioral vulnerability to repeated exposure to restraint stress. Importantly, rescuing the HSF1 deficiency in the neonatal but not the adult hippocampus reversed the aberrant anxiety and depression-like behaviors. These results indicate a crucial role for hippocampal HSF1 in neuronal and behavioral development. Analysis of the molecular mechanisms revealed that HSF1 directly modulates the expression of polysialyltransferase genes, which then modulate polysialic acid-neural cell adhesion molecule (PSA-NCAM) levels in the hippocampus. Enzymatic removal of PSA from the neonatal hippocampus resulted in aberrant behavior during adulthood, similar to that observed in HSF1(-/-) mice. Thus, these results suggest that one role of HSF1 is to control hippocampal PSA-NCAM levels through the transcriptional regulation of polysialyltransferases, a process that might be involved in neuronal and behavioral development in mice.

Roles of heat shock factors in gametogenesis and development

Heat shock factors form a family of transcription factors (four in mammals), which were named according to the first discovery of their activation by heat shock. As a result of the universality and robustness of their response to heat shock, the stress-dependent activation of heat shock factor became a ‘paradigm’: by binding to conserved DNA sequences (heat shock elements), heat shock factors trigger the expression of genes encoding heat shock proteins that function as molecular chaperones, contributing to establish a cytoprotective state to various proteotoxic stress and in several pathological conditions. Besides their roles in the stress response, heat shock factors perform crucial roles during gametogenesis and development in physiological conditions. First, during these process, in stress conditions, they are either proactive for survival or, conversely, for apoptotic process, allowing elimination or, inversely, protection of certain cell populations in a way that prevents the formation of damaged gametes and secure future reproductive success. Second, heat shock factors display subtle interplay in a tissue- and stage-specific manner, in regulating very specific sets of heat shock genes, but also many other genes encoding growth factors or involved in cytoskeletal dynamics. Third, they act not only by their classical transcription factor activities, but are necessary for the establishment of chromatin structure and, likely, genome stability. Finally, in contrast to the heat shock gene paradigm, heat shock elements bound by heat shock factors in developmental process turn out to be extremely dispersed in the genome, which is susceptible to lead to the future definition of ‘developmental heat shock element’.

Roles of heat shock factor 1 and 2 in response to proteasome inhibition: consequence on p53 stability

A single heat shock factor (HSF), mediating the heat shock response, exists from yeast to Drosophila, whereas several related HSFs have been found in mammals. This raises the question of the specific or redundant functions of the different members of the HSF family and in particular of HSF1 and HSF2, which are both ubiquitously expressed. Using immortalized mouse embryonic fibroblasts (iMEFs) derived from wild-type, Hsf1(-/-), Hsf2(-/-) or double-mutant mice, we observed the distinctive behaviors of these mutants with respect to proteasome inhibition. This proteotoxic stress reduces to the same extent the viability of Hsf1(-/-)- and Hsf2(-/-)-deficient cells, but through different underlying mechanisms. Contrary to Hsf2(-/-) cells, Hsf1(-/-) cells are unable to induce pro-survival heat shock protein expression. Conversely, proteasome activity is lower in Hsf2(-/-) cells and the expression of some proteasome subunits, such as Psmb5 and gankyrin, is decreased. As gankyrin is an oncoprotein involved in p53 degradation, we analyzed the status of p53 in HSF-deficient iMEFs and observed that it was strongly stabilized in Hsf2(-/-) cells. This study points a new role for HSF2 in the regulation of protein degradation and suggests that pan-HSF inhibitors could be valuable tools to reduce chemoresistance to proteasome inhibition observed in cancer therapy.

An inducible artificial microRNA system for Chlamydomonas reinhardtii confirms a key role for heat shock factor 1 in regulating thermotolerance

Several RNA silencing strategies employing antisense or inverted repeat constructs have been applied to Chlamydomonas reinhardtii. Problems inherent to these strategies, like off-target effects by unpredictable generation of siRNAs, were solved previously by constructs allowing for routine expression of specific artificial microRNAs (amiRNAs). Yet missing was a routine tool for inducible amiRNA expression, which to establish was the aim of this work. For this, we equipped a recently developed amiRNA expression vector with the NIT1 promoter, which is repressed by ammonium and activated by nitrate. We tested this conditional amiRNA vector with heat shock factor 1 (HSF1) as target. HSF1 transcripts in transformants were already reduced ~2 h after transfer from ammonium to nitrate-containing medium. In contrast, HSF1 protein levels declined only ~8 h after the shift and were strongly reduced after 24 h, suggesting that HSF1 is a stable protein and diluted out by growth. HSF1 levels recovered partly when transformant cells were shifted back to ammonium for 72 h. Transformants developed thermosensitivity only on nitrate and thermosensitivity correlated with strong reduction in HSF1 levels, hence supporting our earlier conclusion that HSF1 is a key regulator for thermotolerance in Chlamydomonas.

A physiological role of heat shock factor 1‐related stress response in the regrowth of atrophied soleus muscle in mice

Hypertrophic stimuli, such as mechanical loading and heat stress, could up‐regulate heat shock proteins (HSPs) in muscle cells. Although it has been proposed that HSPs has several cellular functions, the physiological roles for HSPs induction in skeletal muscles is still unclear. The induction of HSPs expression is regulated by heat shock factor 1 (HSF1), which binds to heat shock elements located on the upstream region of all Hsp genes. In the present study, using the HSF1 knockout (HSF1‐KO) mice, we examined the role of HSPs inthe reloading‐associated regrowth of atrophied soleus muscle. The regrowth was induced by the reloading on soleus following 2‐week‐hindlimb suspension. Increment in the protein content and the up‐regulations of HSPs of soleus muscles in HSF1‐KO mice was smaller than that of wild type animals. Results from this study indicated that the up‐regulation of HSPs may facilitate the protein synthesis in skeletal muscle cells. This study was supported, in part, by Grant‐in‐Aid for Scientific Research (B, 20300218, KG; A, 18200042, TY; S, 19100009, YO) from Japan Society for the Promotion of Science, and Research Grant from KAO Health Science Research (KG).

Positive Role of CCAAT/Enhancer-Binding Protein Homologous Protein, a Transcription Factor Involved in the Endoplasmic Reticulum Stress Response in the Development of Colitis

Although recent reports suggest that the endoplasmic reticulum (ER) stress response is induced in association with the development of inflammatory bowel disease, its role in the pathogenesis of inflammatory bowel disease remains unclear. The CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) is a transcription factor that is involved in the ER stress response, especially ER stress-induced apoptosis. In this study, we found that experimental colitis was ameliorated in CHOP-null mice, suggesting that CHOP exacerbates the development of colitis. The mRNA expression of Mac-1 (CD11b, a positive regulator of macrophage infiltration), Ero-1alpha, and Caspase-11 (a positive regulator of interleukin-1beta production) in the intestine was induced with the development of colitis, and this induction was suppressed in CHOP-null mice. ERO-1alpha is involved in the production of reactive oxygen species (ROS); an increase in ROS production, which is associated with the development of colitis in the intestine, was suppressed in CHOP-null mice. A greater number of apoptotic cells in the intestinal mucosa of wild-type mice were observed to accompany the development of colitis compared with CHOP-null mice, suggesting that up-regulation of CHOP expression exacerbates the development of colitis. Furthermore, this CHOP activity appears to involve various stimulatory mechanisms, such as macrophage infiltration via the induction of Mac-1, ROS production via the induction of ERO-1alpha, interleukin-1beta production via the induction of Caspase-11, and intestinal mucosal cell apoptosis.

Heat shock factor-1 modulates p53 activity in the transcriptional response to DNA damage

Here we define an important role for heat shock factor 1 (HSF1) in the cellular response to genotoxic agents. We demonstrate for the first time that HSF1 can complex with nuclear p53 and that both proteins are co-operatively recruited to p53-responsive genes such as p21. Analysis of natural and synthetic cis elements demonstrates that HSF1 can enhance p53-mediated transcription, whilst depletion of HSF1 reduces the expression of p53-responsive transcripts. We find that HSF1 is required for optimal p21 expression and p53-mediated cell-cycle arrest in response to genotoxins while loss of HSF1 attenuates apoptosis in response to these agents. To explain these novel properties of HSF1 we show that HSF1 can complex with DNA damage kinases ATR and Chk1 to effect p53 phosphorylation in response to DNA damage. Our data reveal HSF1 as a key transcriptional regulator in response to genotoxic compounds widely used in the clinical setting, and suggest that HSF1 will contribute to the efficacy of these agents.

Protective role of HSF1 and HSP70 against gastrointestinal diseases

Purpose: It is well known that heat shock proteins (HSPs) are induced by various stressors in order to confer protection against such stressors. Since stressor-induced tissue damage is involved in various diseases, especially gastrointestinal diseases, it has been thought that HSP-inducers are therapeutically beneficial for these diseases. Indirect lines of evidence suggest that HSPs provide a major protective mechanism against irritant-induced gastric lesions. However, no direct evidence exists to support this notion. On the other hand, inflammatory bowel disease (IBD) involves infiltration of leukocytes into intestinal tissue, resulting in intestinal damage. Pro-inflammatory cytokines and cell adhesion molecules (CAMs) play important roles in this infiltration of leukocytes. The roles of heat shock factor 1 (HSF1, a transcription factor for hsp genes) and heat shock proteins (HSPs) in development of IBD are unclear. In this paper, we reviewed our recent work on the role of HSPs in pathogenesis of gastric lesions and IBD by use of HSF1-null mice and transgenic mice expressing Hsp70.Conclusion: This study provides the first genetic evidence that HSF1 and Hsp70 play a role in protecting against both irritant-induced gastric lesions and IBD-related colitis. The aggravation of irritant-induced gastric lesions in HSF1-null mice is due to their inability to up-regulate Hsp70, leading to apoptosis. On the other hand, this protective role of Hsp70 against colitis seems to involve various mechanisms, such as suppression of expression of pro-inflammatory cytokines and CAMs, and cell death.

Cellular and Molecular Mechanisms of Heat Stress-Induced Up-Regulation of Occludin Protein Expression: Regulatory Role of Heat Shock Factor-1

The heat stress (HS)-induced increase in occludin protein expression has been postulated to be a protective response against HS-induced disruption of the intestinal epithelial tight junction barrier. The aim of this study was to elucidate the cellular and molecular processes that mediate the HS-induced up-regulation of occludin expression in Caco-2 cells. Exposure to HS (39 degrees C or 41 degrees C) resulted in increased expression of occludin protein; this was preceded by an increase in occludin mRNA transcription and promoter activity. HS-induced activation of heat shock factor-1 (HSF-1) resulted in cytoplasmic-to-nuclear translocation of HSF-1 and binding to its binding motif in the occludin promoter region. HSF-1 activation was associated with an increase in occludin promoter activity, mRNA transcription, and protein expression; which were abolished by the HSF-1 inhibitor quercetin. Targeted HSF-1 knock-down by siRNA transfection inhibited the HSF-1-induced increase in occulin expression and junctional localization of occulin protein. Site-directed mutagenesis of the HSF-1 binding motif in the occludin promoter region inhibited HS-induced binding of HSF-1 to the occludin promoter region and subsequent promoter activity. In conclusion, our data show for the first time that the HS-induced increase in occludin protein expression is mediated by HSF-1 activation and subsequent binding of HSF-1 to the occludin promoter, which initiates a series of molecular and cellular events culminating in increased junctional localization of occludin protein.