Heat Shock Factor Protein Research Articles

Direct activation of HSF1 by macromolecular crowding and misfolded proteins.

Stress responses play a vital role in cellular survival against environmental challenges, often exploited by cancer cells to proliferate, counteract genomic instability, and resist therapeutic stress. Heat shock factor protein 1 (HSF1), a central transcription factor in stress response pathways, exhibits markedly elevated activity in cancer. Despite extensive research into the transcriptional role of HSF1, the mechanisms underlying its activation remain elusive. Upon exposure to conditions that induce protein damage, monomeric HSF1 undergoes rapid conformational changes and assembles into trimers, a key step for DNA binding and transactivation of target genes. This study investigates the role of HSF1 as a sensor of proteotoxic stress conditions. Our findings reveal that purified HSF1 maintains a stable monomeric conformation independent of molecular chaperones in vitro. Moreover, while it is known that heat stress triggers HSF1 trimerization, a notable increase in trimerization and DNA binding was observed in the presence of protein-based crowders. Conditions inducing protein misfolding and increased protein crowding in cells directly trigger HSF1 trimerization. In contrast, proteosynthesis inhibition, by reducing denatured proteins in the cell, prevents HSF1 activation. Surprisingly, HSF1 remains activated under proteotoxic stress conditions even when bound to Hsp70 and Hsp90. This finding suggests that the negative feedback regulation between HSF1 and chaperones is not directly driven by their interaction but is realized indirectly through chaperone-mediated restoration of cytoplasmic proteostasis. In summary, our study suggests that HSF1 serves as a molecular crowding sensor, trimerizing to initiate protective responses that enhance chaperone activities to restore homeostasis.

Discovery of Disubstituted Carboranes as Inhibitors of Heat Shock Protein 90-Heat Shock Factor 1 Interaction.

Efficient synthesis of disubstituted para- and ortho-carboranes (2 and 3, respectively) was achieved. Among the compounds synthesized, 3e showed potent suppression of hypoxia-inducible factor 1 (HIF-1) transcriptional activity under hypoxia by a cell-based reporter gene assay. Detailed mechanism-of-action studies revealed that 3e reduced the stability of heat shock protein (HSP) 90 client proteins such as CDK4, AKT, and cyclin D1 by inhibiting HSP90 chaperone activity but did not induce a heat shock response (HSR), which may cause drug resistance. Furthermore, 3e inhibited the interaction between HSP90 and heat shock factor 1 (HSF1), resulting in reducing HSF1 protein stability and thereby suppressing the transcription of heat shock proteins.

Abstract 4319: HSF1 inhibition induces pancreatic ductal adenocarcinoma autophagy through JNK

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related mortality, with a 5-year survival rate of 12%. Chemoresistance due to autophagy, a natural process of proteotoxic stress response, is the most common chemotherapy challenge for pancreatic cancer. Autophagy, which maintains cellular homeostasis by removing damaged or unnecessary proteins and organelles, is a self-degradation process regulated by the mammalian target of rapamycin complex (mTORC1). Excessive levels of autophagy can be detrimental and lead to cell death. Nevertheless, this dual function of autophagy in regulating pancreatic cancer remains poorly understood. It has been reported that heat shock factor 1 (HSF1), a critical pro-oncogenic transcription factor in the proteotoxic stress response, inhibits c-Jun N-terminal kinase 1/2 (JNK1/2) signaling pathway to maintain mTORC1 activity and control cell size. HSF1 is highly expressed in pancreatic tumors, and inhibition of HSF1 via small molecules enhances the chemotherapy drug effect on PDAC programmed cell death. However, the PDAC autophagy response in HSF1 inhibition is currently not fully understood. Objective: The objective of this study is to investigate the molecular mechanism of HSF1 inhibition in PDAC autophagy. Methods and Results: In human PDAC cells, Western blotting analysis revealed that HSF1 inhibition via small molecular including CCT361814/NXP800, an HSF1 inhibitor in phase I clinical trials, increased autophagy marker microtubule-associated protein 1A/1B-light chain 3 (LC3) lipidation and decreased expression of phosphorylation of HSF1 at Ser326 and total HSF1 protein, conversely using HSF1 overexpression model reversed this effect. Inhibition of autophagy via 3-methyladenine reversed HSF1 inhibition-induced autophagy. Results from transmission electron microscopy and GFP-LC3 reporter revealed that HSF1 inhibition induced autophagosome formation and accumulation. Furthermore, HSF1 inhibition induced phosphorylation of JNK1/2 at Thr183/Tyr185 and decreased mTORC1 activity, while knockdown JNK1/2 diminished HSF1 inhibition-induced LC3 lipidation. The combination of chemotherapy drugs with HSF1 inhibitors decreased PDAC cell viability. Additionally, blocking autophagy increased HSF1 inhibition-induced apoptosis. Conclusions: In conclusion, JNK1/2 is involved in HSF1 inhibition-induced PDAC autophagy, targeting autophagy could unveil a novel treatment strategy for pancreatic cancer through targeted HSF1 inhibition. Citation Format: Shruti Ghai, Alex Young, Kuo-Hui Su. HSF1 inhibition induces pancreatic ductal adenocarcinoma autophagy through JNK [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4319.

Abstract B031: The PLK1 inhibitor Volasertib as a treatment for HSF1 and MYC coamplified ovarian cancer

Abstract Ovarian cancer is a disease that afflicts 1 in 78 women. This disease only has a 5-year relative survival rate of 49% due to the fact that the first line chemotherapy for this disease, platinum-based chemotherapy with taxanes, has an 80% recurrence rate. To improve the treatment for this disease, it would be wise to find biomarkers of sensitivity to other drugs. Using publicly available data from The Cancer Genome Project, our lab has shown that 36% of High Grade Serous Ovarian Cancers (HGSOC) have gene amplifications for both Cellular Myelocytomatosis (MYC) and Heat Shock Factor 1 (HSF1) genes. These genes encode for transcription factors and are known to contribute to malignant disease in multiple cancer types, including ovarian cancer. Evidence indicates that MYC and HSF1 are both essential to HGSOC cells with co-amplification of these genes, that MYC and HSF1 transcriptional activities are highly correlated in patient tumors, and the two transcription factors have a protein-protein interaction, all suggesting these two transcription factors cooperate to drive this subset of HGSOC tumors. Many drugs have been developed to inhibit MYC or HSF1 though they have not shown success in clinical development. We aimed to decrease the activity of these proteins by targeting polo-like kinase 1 (PLK1), a kinase that phosphorylates and regulates both MYC and HSF1 proteins. Active PLK1 levels were highly correlated with MYC and HSF1 protein levels and inhibition of PLK1 with volasertib reduced MYC and HSF1 protein levels and transcriptional activity. Furthermore, volasertib was found to have 155-fold greater potency in HGSOC cells with MYC-HSF1 co-amplification compared to cells without these genes amplified. The increased potency of volasertib in MYC-HSF1 co-amplified cells were also observed in clonogenic growth and spheroid models. Together, these data indicate that MYC and HSF1 cooperate in HGSOC when these genes are amplified and targeting a common kinase between them in PLK1 disrupts this cooperation. Furthermore, MYC and HSF1 co-amplification may serve as a biomarker for volasertib through a personalized medicine approach to treat HGSOC patients. Citation Format: Matthew O'Malley, Imade W. Williams, Bobby Walker, Haimanti Ray, Kenneth P. Nephew, Richard Carpenter. The PLK1 inhibitor Volasertib as a treatment for HSF1 and MYC coamplified ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B031.

Association between heat shock factor protein 4 methylation and colorectal cancer risk and potential molecular mechanisms: A bioinformatics study.

We previously demonstrated that heat shock factor protein 4 (HSF4) facilitates colorectal cancer (CRC) progression. DNA methylation, a major modifier of gene expression and stability, is involved in CRC development and outcome. To investigate the correlation between HSF4 methylation and CRC risk, and to uncover the underlying molecular mechanisms. Differences in β values of HSF4 methylation loci in multiple malignancies and their correlation with HSF4 mRNA expression were analyzed based on Shiny Methylation Analysis Resource Tool. HSF4 methylation-related genes were identified by LinkedOmics in CRC, and Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed. Protein-protein interaction network of HSF4 methylation-related genes was constructed by String database and MCODE algorithm. A total of 19 CpG methylation loci were identified in HSF4, and their β values were significantly increased in CRC tissues and exhibited a positive correlation with HSF4 mRNA expression. Unfortunately, the prognostic and diagnostic performance of these CpG loci in CRC patients was mediocre. In CRC, there were 1694 HSF4 methylation-related genes; 1468 of which displayed positive and 226 negative associations, and they were involved in regulating phenotypes such as immune, inflammatory, and metabolic reprogramming. EGFR, RELA, STAT3, FCGR3A, POLR2K, and AXIN1 are hub genes among the HSF4 methylation-related genes. HSF4 is highly methylated in CRC, but there is no significant correlation between it and the prognosis and diagnosis of CRC. HSF4 methylation may serve as one of the ways in which HSF4 mediates the CRC process.

Overexpression of GmPIF4b affects morpho-physiological traits to reduce heat-induced grain loss in soybean

Heat waves associated with climate change seriously threaten crop productivity. Crop seed yield depends on the success of reproduction. However, reproductive development is most vulnerable to heat stress conditions. Perception of heat and its conversion into cellular signals is a complex process. The basic helix loop helix (bHLH) transcription factor, Phytochrome Interacting Factor 4 (PIF4), plays a significant role in this process. However, studies on PIF4- mediated impacts on crop grain yield at a higher temperature are lacking. We investigated the overexpression of GmPIF4b in soybean to alleviate heat-induced damage and yield using a transgenic approach. Our results showed that under high-temperature conditions (38°C/28°C), overexpressing soybeans plants had higher chlorophyll a and b, and lower proline accumulation compared to WT. Further, overexpression of GmPIF4b improved pollen viability under heat stress and reduced heat-induced structural abnormalities in the male and female reproductive organs. Consequently, the transgenic plants produced higher pods and seeds per plant at high temperatures. Quantitative RT-PCR analysis showed that the overexpressing GmPIF4b soybeans had higher transcripts of heat shock factor, GmHSF-34, and heat-shock protein, GmHSP90A2. Collectively, our results suggest that GmPIF4b regulates multiple morpho-physiological traits for better yield under warmer climatic conditions.

HSF1 is a novel prognostic biomarker in high-risk prostate cancer that correlates with ferroptosis

BackgroundProstate cancer (PC) is the most common cancer in older men in Europe and the United States and has the second highest death rate among male cancers. The transcription of heat shock proteins by Heat shock factor 1 (HSF1) is known to regulate cell growth and stress. Nevertheless, the impact of HSF1 on ferroptosis in PC through heat shock protein 10 (HSPE1) remains unexplored.MethodsThis study employed a range of analytical techniques, including proteomics sequencing, LC–MS/MS, CHIP-qPCR, Western blotting, immunohisto -chemistry, JC-1, CKK-8, MDA, and ROS assays. Bioinformatics analysis was performed using the UALCAN,GEPIA, PCaDB and Metascape platforms.ResultsCompared with levels observed in tumor-adjacent tissue, the levels of proteins associated with fatty acids, amino acids and the oxidative phosphorylation metabolic pathway were significantly upregulated in high-risk PC tissue (Gleason score ≥ 8). HSF1 mRNA and protein levels in high-risk PC tissues were significantly higher than those observed in medium-risk PC (Gleason score = 7) and low-risk PC (Gleason score ≤ 6) tissues. ssGSEA showed that HSF1 was involved in the proliferation and anti-apoptotic processes of PC. Further bioinformatics analysis showed that HSF1 potentially affects the mitochondrial oxidative phosphorylation (OXPHOS) system by targeting HSPE1. In addition, HSF1 alleviates ROS and MDA levels to enhance the resistance of prostate cancer cells to ferroptosis by regulating HSPE1 in vitro, and HSF1 knockout promotes the susceptibility of PC to RSL3 treatment by increasing ferroptosis in vivo.ConclusionCollectively, our findings suggest that HSF1 exerts a significant influence on PC. HSF1 may represent a promising biomarker for identifying high-risk PC, and the elimination of HSF1 could potentially enhance the therapeutic effectiveness of RSL3.

Interaction between transcriptional activator BRI1-EMS-SUPPRESSOR 1 and HSPs regulates heat stress tolerance in pepper

Climate change is predicted to stymie agricultural productivity in the future. To mitigate these detrimental consequences, traditional breeding and genetic engineering of environmentally tolerant plants are necessary. The discovery of genes related to stress tolerance is crucial to achieving this goal. In this study, we conducted transcriptome analyses of heat-tolerant and heat-sensitive pepper species to identify the genetic elements governing heat-stress tolerance in pepper plants. Using WGCNA (Weighted Gene Co-expression Network Analysis), we created co-expression networks from the transcriptome data and identified a heat-related module (ME.cyan), in which the BES1 transcription factor, CcBES1 (CcBES1–4), plays a central role in the heat tolerance mechanism in pepper. We successfully identified and functionally characterized the BES1 gene from the pepper. CcBES1 is predominantly a transcriptional activator encoded by a nucleus-localized protein. We generated CcBES1-silenced pepper lines and conducted heat stress assays in vitro. Dwarf-specific phenotypes and heat stress susceptibility were observed in the CcBES1-silenced line. Moreover, most of the Heat shock factor proteins (HSPs) expression in CcBES1-silenced lines were consistent with the ME.cyan module co-expression network genes. Yeast one-hybrid results revealed that CcBES1 directly binds to the putative promotor regions of the HSPs, indicating that CcBES1 controls co-expressed HSPs to govern heat stress tolerance. The findings of the present study demonstrate approaches to trigger substantial networks augmented for thermotolerance, laying the foundation for future studies into the management of heat stress regulation.

HSF1 Alleviates Brain Injury by Inhibiting NLRP3-Induced Pyroptosis in a Sepsis Model.

Sepsis, which could cause a systemic inflammatory response, is a life-threatening disease with a high morbidity and mortality rate. There is evidence that brain injury may be related to severe systemic infection induced by sepsis. The brain injury caused by sepsis could increase the risk of mortality in septic patients, which seriously affects the septic patient's prognosis of survival. Although there remains a focus on sepsis research, clinical measures to prevent and treat brain injury in sepsis are not yet available, and the high mortality rate is still a big health burden. Therefore, it is necessary to investigate the new molecules or regulated pathways that can effectively inhibit the progress of sepsis. NLR family pyrin domain-containing 3 (NLRP3) increased in the procession of sepsis and functioned as the key regulator of pyroptosis. Heat shock factor 1 (HSF1) can protect organs from multiorgan dysfunction syndrome induced by lipopolysaccharides in mice, and NLRP3 could be inhibited by HSF1 in many organs. However, whether HSF1 regulated NLRP3 in sepsis-induced brain injury, as well as the detailed mechanism of HSF1 in brain injury, remains unknown in the sepsis model. In this research, we try to explore the relationship between HSF1 and NLRP3 in a sepsis model and try to reveal the mechanism of HSF1 inhibiting the process of brain injury. In this study, we used wild-type mice and hsf1 -/- mice for in vivo research and PC12 cells for in vitro research. Real-time PCR and Western blot were used to analyze the expression of HSF1, NLRP3, cytokines, and pyrolytic proteins. EthD-III staining was chosen to detect the pyroptosis of the hippocampus and PC12 cells. The results showed that HSF1 is negatively related to pyroptosis. The pyroptosis in cells of brain tissue was significantly increased in the hsf1 -/- mouse model compared to hsf1 +/+ mice. In PC12 cells, hsf1 siRNA can upregulate pyroptosis while HSF1-transfected plasmid could inhibit the pyroptosis. HSF1 could negatively regulate the NLRP3 pathway in PC12 cells, while hsf1 siRNA enhanced the pyroptosis in PC12 cells, which could be reversed by nlrp3 siRNA. These results imply that HSF1 could alleviate sepsis-induced brain injury by inhibiting pyroptosis through the NLRP3-dependent pathway in brain tissue and PC12 cells, suggesting HSF1 as a potential molecular target for treating brain injury in sepsis clinical studies.

HSF1 expression in tumor-associated macrophages promotes tumor cell proliferation and indicates poor prognosis in esophageal squamous cell carcinoma.

Tumor-associated macrophages (TAMs), are crucial for the survival and development of tumor cells. Heat shock factor 1 (HSF1) is a potent, complex carcinogenesis modulator, and esophageal cancer (EC) patients have a bad prognosis when HSF1 is highly expressed. HSF1's clinical importance and biological role in TAMs are still unknown. The HSF1 expression profile and patient survival information were analyzed from the TCGA database. The infiltration of different types of immune cells in EC was evaluated based on HSF1 gene expression by Sangerbox 3.0. Immunochemistry was employed to assess HSF1 protein expression in 134 individuals with esophageal squamous cell carcinoma (ESCC), proceeded by association with clinicopathological variables. The role of macrophage-driven HSF1 were observed using HSF1-knockdown THP1 cells. High level of HSF1 have a poorer prognosis in individuals with EC. The expressing level of HSF1 was positively related to infiltration of M2 macrophages (P < 0.05). The expression of HSF1 in macrophages was an independent factor for DFS (P = 0.002) and OS (P = 0.002) in ESCC cases. HSF1 was up-regulated in IL-4 stimulation THP1 cells in a time-dependent manner. Under the heat stimulation condition, THP1-derived macrophages were more sensitive than tumor cells. Compared to IL-4 induced-THP1 cells control, the HSF1 knockdown in THP1 cell inhibited the growth and proliferation of ESCC cells. The up-regulation of HSF1 was more rapid and could affect the proliferation of tumor cells in IL4-induced macrophages. The expression of HSF1 in TAMs can also serve as a marker for ESCC prognosis.

RNA Sequencing indicates gene expression changes in Silene sendtneri seeds after seed priming with silicic acid

To improve our understanding of the molecular mechanisms underlaying seed priming, RNA transcriptome analysis was performed using primed and non-primed seeds of Silene sendtneri. Seed priming was performed by submergence in 1% silicic acid for 24h at 4°C, followed by rinsing with sterilised water and desiccation to original moisture content. Silene sendtneri is a species with no sequenced genome and annotation of de novo assembly of transcriptome was done against several species. Gene ontology (GO) analysis indicated that genes related to heavy metal transporters and heat shock proteins are differentially expressed after priming with silicic acid. Within these gene categories, genes such as heavy metal-associated isoprenylated plant protein 26-like (log2fold -8.79) were downregulated, while others such as heavy metal ATPase 5 (log2fold 6.46), heat shock factor protein HSF30-like isoform X1 (log2fold 5.98) were upregulated.

Alleviating effect of exogenous melatonin and calcium on the peroxidation damages of cucumber under high temperature stress

To explore whether there is an interaction between melatonin (MT) and calcium (Ca2+) in regulating heat tolerance of plants, we analyzed the response of endogenous MT and Ca2+ to heat stress, and examined the effect of MT and Ca2+ on the reactive oxygen (ROS) accumulation, antioxidant system, and transcripts of heat shock factor (HSF) and heat shock proteins (HSPs) of cucumber seedlings under high temperature stress. Seedlings were foliar sprayed with 100 μmol·L-1 MT, 10 mmol·L-1 CaCl2, 3 mmol·L-1 ethylene glycol tetraacetic acid (EGTA, Ca2+ chelating agent) +100 μmol·L-1 MT, 0.05 mmol·L-1 chlorpromazine (calmodulin antagonist, CPZ) +100 μmol·L-1 MT, 100 μmol·L-1 p-chlorophenylalanine (p-CPA, inhibitor of MT) +10 mmol·L-1 CaCl2 or deionized water (H2O), respectively. The results showed that both endogenous MT and Ca2+ in cucumber seedlings were induced by high temperature stress. The seedlings treated with exogenous MT showed significant increases in the mRNA expression of calmodulin (CaM), calcium-dependent protein kinase (CDPK5), calcineurin B-like protein (CBL3) and CBL interacting protein kinase (CIPK2) compared with the control at normal temperature. The mRNA levels of tryptophane decarboxylase (TDC), 5-hydroxytryptamine-N-acetyltransferase (SNAT) and N-acetyl-5-hydroxytryptamine methyltransferase (ASMT), key genes of MT biosynthesis and endogenous MT content were also induced by Ca2+ in cucumber seedlings. Exogenous MT and CaCl2 alleviated the heat-induced oxidative damage through increasing antioxidant ability, reducing the accumulation of reactive oxygen species (ROS), and upregulating the mRNA abundances of HSF7, HSP70.1 and HSP70.11, as evidenced by mild thermal damage symptoms, lower heat injury index and electrolyte leakage under heat stress. The positive effect of MT-induced antioxidant capacity and mRNA expression of HSPs was removed by adding EGTA and CPZ in stressed seedlings. Similarly, the mitigating role of Ca2+ in the peroxidation damage to high temperature stress was reversed by p-CPA. These results suggested that both MT and Ca2+ could induce heat tolerance of cucumber seedlings, which had crosstalk in the process of heat stress signal transduction.

Melatonin ameliorates lipopolysaccharide induced brain inflammation through modulation of oxidative status and diminution of cytokine rush in Danio rerio

Lipopolysaccharide (LPS) is known to induce inflammation and immunonomodulation in a piscine model of Danio rerio. Present study aimed to explore the ability of melatonin in attenuating LPS-induced oxidative damages using this model. In LPS-exposed fish, activation of stress marker MDA was observed in brain with corresponding augmentation of multiple pro-inflammatory cytokines (IL1β, IL6, IL10 and TNFα). In addition, it also showed marked increase in the levels of heat shock factor (HSF) and heat shock proteins (HSPs) in association with transcription factors (NF-kB and NRF2) and mitogen-activated protein kinases (MAPKs). The changes in the levels of these mediators are highly correlated with the induction of pro-inflammatory cytokines. In melatonin-treated fishes, significant amelioration of oxidative stress was observed with reduced levels of MDA and pro-inflammatory cytokines. Melatonin also modulated expression of HSPs that facilitated the brain to overcome inflammation-induced stress by directly initiating NFkB/NRF2 translocation. In summary, melatonin effectively functions to reduce stress induced inflammatory signalling through modulation of oxidative stress and protects the brain from the neuropathological insult.

Identification, expression, and functional analysis of Hsf and Hsp20 gene families in Brachypodium distachyon under heat stress.

BackgroundThe heat shock factor (Hsf) and small heat shock protein (sHsp, also called Hsp20) complex has been identified as a primary component in the protection of plant cells from ubiquitous stresses, particularly heat stress. Our study aimed to characterize and analyze the Hsf and Hsp genes in Brachypodium distachyon, an annual temperate grass and model plant in cereal and grass studies.ResultsWe identified 24 Hsf and 18 Hsp20 genes in B. distachyon and explored their evolution in gene organization, sequence features, chromosomal localization, and gene duplication. Our phylogenetic analysis showed that BdHsfs could be divided into three categories and BdHsp20s into ten subfamilies. Further analysis showed that the 3’UTR length of BdHsp20 genes had a negative relationship with their expression under heat stress. Expression analyses indicated that BdHsp20s and BdHsfs were strongly and rapidly induced by high-temperature treatment. Additionally, we constructed a complex regulatory network based on their expression patterns under heat stress. Morphological analysis suggested that the overexpression of five BdHsp20 genes enhanced the seed germination rate and decreased cell death under high temperatures.ConclusionUltimately, our study provided important evolutionary and functional characterizations for future research on the regulatory mechanisms of BdHsp20s and BdHsfs in herbaceous plants under environmental stress.

Heat Shock Factor 1 Prevents Age-Related Hearing Loss by Decreasing Endoplasmic Reticulum Stress.

Endoplasmic reticulum (ER) stress is a common stress factor during the aging process. Heat shock factor 1 (HSF1) plays a critical role in ER stress; however, its exact function in age-related hearing loss (ARHL) has not been fully elucidated. The purpose of the present study was to identify the role of HSF1 in ARHL. In this study, we demonstrated that the loss of inner and outer hair cells and their supporting cells was predominant in the high-frequency region (basal turn, 32 kHz) in ARHL cochleae. In the aging cochlea, levels of the ER stress marker proteins p-eIF2α and CHOP increased as HSF1 protein levels decreased. The levels of various heat shock proteins (HSPs) also decreased, including HSP70 and HSP40, which were markedly downregulated, and the expression levels of Bax and cleaved caspase-3 apoptosis-related proteins were increased. However, HSF1 overexpression showed significant hearing protection effects in the high-frequency region (basal turn, 32 kHz) by decreasing CHOP and cleaved caspase-3 and increasing the HSP40 and HSP70 proteins. These findings were confirmed by HSF1 functional studies using an auditory cell model. Therefore, we propose that HSF1 can function as a mediator to prevent ARHL by decreasing ER stress-dependent apoptosis in the aging cochlea.

BSCI-05. ABL2-HSF1-E2F signaling axis promotes lung adenocarcinoma brain metastasis

Brain metastases are the most common intracranial tumors in adults and are associated with increased patient morbidity and mortality. Limited therapeutic options are currently available for the treatment of brain metastasis. We have identified an actionable signaling pathway utilized by metastatic tumor cells whereby the transcriptional regulator Heat Shock Factor 1 (HSF1) drives a transcriptional program, divergent from its canonical role as the master regulator of the heat shock response, leading to enhanced expression of a subset of E2F transcription factor family gene targets. We showed that HSF1 is required for survival and outgrowth by metastatic lung cancer cells in the brain parenchyma. Unexpectedly, we identified the ABL2 tyrosine kinase as an upstream regulator of HSF1 protein expression, and showed that the Src-homology 3 (SH3) domain of ABL2 directly interacts with HSF1 protein at a non-canonical, proline-independent SH3 interaction motif. Importantly, knockdown of ABL2 impairs expression of HSF1 protein and HSF1-E2F transcriptional gene targets. Notably, we found that pharmacologic inhibition of the ABL kinases using selective ABL allosteric inhibitors, but not ATP-competitive inhibitors, ablates the physical interaction between ABL2 and HSF1, leading to markedly decreased expression of HSF1, E2F1 and E2F8 proteins in brain-metastatic lung cancer cells, and depletion of HSF1-E2F transcriptional targets. These findings highlight potential differences affecting intra- and inter-molecular protein-protein interactions induced by allosteric versus ATP-competitive kinase inhibitors that have important therapeutic implications. Importantly, the targetable nature of the ABL2-HSF1-E2F signaling network identifies ABL allosteric inhibitors as a potentially effective therapy for the treatment of metastatic lung cancers characterized by high expression of HSF1.

Augmentation of the heat shock axis during exceptional longevity in Ames dwarf mice

How the heat shock axis, repair pathways, and proteostasis impact the rate of aging is not fully understood. Recent reports indicate that normal aging leads to a 50% change in several regulatory elements of the heat shock axis. Most notably is the age-dependent enhancement of inhibitory signals associated with accumulated heat shock proteins and hyper-acetylation associated with marked attenuation of heat shock factor 1 (HSF1)–DNA binding activity. Because exceptional longevity is associated with increased resistance to stress, this study evaluated regulatory check points of the heat shock axis in liver extracts from 12 months and 24 months long-lived Ames dwarf mice and compared these findings with aging wild-type mice. This analysis showed that 12M dwarf and wild-type mice have comparable stress responses, whereas old dwarf mice, unlike old wild-type mice, preserve and enhance activating elements of the heat shock axis. Old dwarf mice thwart negative regulation of the heat shock axis typically observed in usual aging such as noted in HSF1 phosphorylation at Ser307 residue, acetylation within its DNA binding domain, and reduction in proteins that attenuate HSF1–DNA binding. Unlike usual aging, dwarf HSF1 protein and mRNA levels increase with age and further enhance by stress. Together these observations suggest that exceptional longevity is associated with compensatory and enhanced HSF1 regulation as an adaptation to age-dependent forces that otherwise downregulate the heat shock axis.

GWAS identified candidate variants and genes associated with acute heat tolerance of large yellow croaker

Heat tolerance is an important ability for fish to cope with increased temperature. Large yellow croaker (Larimichthys crocea) is one of the most economically important mariculture fish in China. However, the sustainable development of large yellow croaker aquaculture has long been hampered by extremely high temperature in summer, which is further intensified by the ever-increased global warming. To relieve mass death caused by extreme heat events, it is essential to improve the heat tolerance of this species, which will benefit from a comprehensive understanding of the genetic basis underlying heat tolerance. Here, using a 650 K high density SNP array, we conducted a genome-wide association study (GWAS) on acute heat tolerance (AHT) of large yellow croaker. As a result, 5 significant single nucleotide polymorphisms (SNPs) on 4 chromosomes were identified, indicating the polygenic control characteristics of this trait. A clear peak on Chr19 was found and most of the nearby genes showed differential expression during heat stress, indicating the potential relevance of this region with AHT. Based on the 5 identified SNPs, we predicted 30 candidate genes including heat shock factor protein 1, DnaJ homolog subfamily B member 4, protein Hikeshi, protein disulfide-isomerase A3 etc. Further comparative and validation analysis suggested the importance of blood vessel regulation, heat shock response and endoplasmic reticulum stress response in mediating the interindividual AHT variation of large yellow croaker. These results provide insights into the genetic basis of AHT in fish and will be helpful for heat-tolerance improvement of large yellow croaker by marker-assisted selection.

The forkhead box transcription factor FoxP4 regulates thermogenic programs in adipocytes

Forkhead box transcription factors have been shown to be involved in various developmental and differentiation processes. In particular, members of the FoxP family have been previously characterized in depth for their participation in the regulation of lung and neuronal cell differentiation and T-cell development and function; however, their role in adipocyte functionality has not yet been investigated. Here, we report for the first time that Forkhead box P4 (FoxP4) is expressed at high levels in subcutaneous fat depots and mature thermogenic adipocytes. Through molecular and gene expression analyses, we revealed that FoxP4 is induced in response to thermogenic stimuli, both in vivo and in isolated cells, and is regulated directly by the heat shock factor protein 1 through a heat shock response element identified in the proximal promoter region of FoxP4. Further detailed analysis involving chromatin immunoprecipitation and luciferase assays demonstrated that FoxP4 directly controls the levels of uncoupling protein 1, a key regulator of thermogenesis that uncouples fatty acid oxidation from ATP production. In addition, through our gain-of-function and loss-of-function studies, we showed that FoxP4 regulates the expression of a number of classic brown and beige fat genes and affects oxygen consumption in isolated adipocytes. Overall, our data demonstrate for the first time the novel role of FoxP4 in the regulation of thermogenic adipocyte functionality.

Comparative Transcriptomics Analysis and Functional Study Reveal Important Role of High-Temperature Stress Response Gene GmHSFA2 During Flower Bud Development of CMS-Based F1 in Soybean.

High-temperature (HT) is one of the most important environmental factors that negatively impact the yield of some soybean cytoplasmic male sterility (CMS)-based hybrid (F1) combinations. The response of soybean to HT, especially at the male organ development stage, is poorly understood. To investigate the molecular mechanisms of the response from soybean CMS-based F1 male organ to HT, a detailed transcriptomics analysis was performed during flower bud development of soybean HT-tolerant and HT-sensitive CMS-based F1 combinations (NF1 and YF1) under normal-temperature and HT conditions. Obvious HT damage was observed by subjecting YF1 with HT, such as indehiscent anthers and decreased pollen fertility, whereas the male fertility of NF1 was normal. In total, 8,784 differentially expressed genes (DEGs) were found to respond to HT stress, which were mainly associated with anther/pollen wall development, carbohydrate metabolism and sugar transport, and auxin signaling. The quantitative real-time PCR (qRT-PCR) analysis and substance content detection also revealed that HT caused male fertility defects in YF1 by altering pectin metabolism, auxin, and sugar signaling pathways. Most importantly, the sugar signaling-PIF-auxin signaling pathway may underlie the instability of male fertility in YF1 under HT. Furthermore, HT induced the expression of heat shock factor (HSF) and heat shock protein (HSP) gene families. Overexpression of GmHSFA2 in Arabidopsis can promote the expression of HT protective genes (such as HSP20) by binding to the HSE motifs in their promoters, so as to improve the HT tolerance during flowering. Our results indicated that GmHSFA2 acted as a positive regulator, conferring HT tolerance improvement in soybean CMS-based F1. GmHSFA2 may be directly involved in the activation of male fertility protection mechanism in the soybean CMS-based F1 under HT stress.