HSF1 Expression Research Articles

BAG3 expression and sarcomere localization in the human heart are linked to HSF-1 and are differentially affected by sex and disease

Mutations to the sarcomere-localized cochaperone protein Bcl2-associated athanogene 3 (BAG3) are associated with dilated cardiomyopathy (DCM) and display greater penetrance in male patients. Decreased protein expression of BAG3 is also associated with nongenetic heart failure; however, the factors regulating cardiac BAG3 expression are unknown. Using left ventricular (LV) tissue from nonfailing and DCM human samples, we found that whole LV BAG3 expression was not significantly impacted by DCM or sex; however, myofilament localized BAG3 was significantly decreased in males with DCM. Females with DCM displayed no changes in BAG3 compared with nonfailing. This sex difference appears to be estrogen independent, as estrogen treatment in ovariectomized female rats had no impact on BAG3 expression. BAG3 gene expression in noncardiac cells is primarily regulated by the heat shock transcription factor-1 (HSF-1). We show whole LV HSF-1 expression and nuclear localized/active HSF-1 each displayed a striking positive correlation with whole LV BAG3 expression. We further found that HSF-1 localizes to the sarcomere Z-disc in cardiomyocytes and that this myofilament-associated HSF-1 pool decreases in heart failure. The decrease of HSF-1 was more pronounced in male patients and tightly correlated with myofilament BAG3 expression. Together our findings indicate that cardiac BAG3 expression and myofilament localization are differentially impacted by sex and disease and are linked to HSF-1.NEW & NOTEWORTHY Myofilament BAG3 expression decreases in male patients with nonischemic DCM but is preserved in female patients with DCM. BAG3 expression in the human heart is tightly linked to HSF-1 expression and nuclear translocation. HSF-1 localizes to the sarcomere Z-disc in the human heart. HSF-1 expression in the myofilament fraction decreases in male patients with DCM and positively correlates with myofilament BAG3.

The heat shock response, determined by QuantiGene multiplex, is impaired in HD mouse models and not caused by HSF1 reduction

Huntington’s disease (HD) is a devastating neurodegenerative disorder, caused by a CAG/polyglutamine repeat expansion, that results in the aggregation of the huntingtin protein, culminating in the deposition of inclusion bodies in HD patient brains. We have previously shown that the heat shock response becomes impaired with disease progression in mouse models of HD. The disruption of this inducible arm of the proteostasis network is likely to exacerbate the pathogenesis of this protein-folding disease. To allow a rapid and more comprehensive analysis of the heat shock response, we have developed, and validated, a 16-plex QuantiGene assay that allows the expression of Hsf1 and nine heat shock genes, to be measured directly, and simultaneously, from mouse tissue. We used this QuantiGene assay to show that, following pharmacological activation in vivo, the heat shock response impairment in tibialis anterior, brain hemispheres and striatum was comparable between zQ175 and R6/2 mice. In contrast, although a heat shock impairment could be detected in R6/2 cortex, this was not apparent in the cortex from zQ175 mice. Whilst the mechanism underlying this impairment remains unknown, our data indicated that it is not caused by a reduction in HSF1 levels, as had been reported.

A Point Mutation in IKAROS ZF1 Causes a B Cell Deficiency in Mice.

IKZF1 (IKAROS) is essential for normal lymphopoiesis in both humans and mice. Previous Ikzf1 mouse models have demonstrated the dual role for IKZF1 in both B and T cell development and have indicated differential requirements of each zinc finger. Furthermore, mutations in IKZF1 are known to cause common variable immunodeficiency in patients characterized by a loss of B cells and reduced Ab production. Through N-ethyl-N-nitrosourea mutagenesis, we have discovered a novel Ikzf1 mutant mouse with a missense mutation (L132P) in zinc finger 1 (ZF1) located in the DNA binding domain. Unlike other previously reported murine Ikzf1 mutations, this L132P point mutation (Ikzf1L132P ) conserves overall protein expression and has a B cell-specific phenotype with no effect on T cell development, indicating that ZF1 is not required for T cells. Mice have reduced Ab responses to immunization and show a progressive loss of serum Igs compared with wild-type littermates. IKZF1L132P overexpressed in NIH3T3 or HEK293T cells failed to localize to pericentromeric heterochromatin and bind target DNA sequences. Coexpression of wild-type and mutant IKZF1, however, allows for localization to pericentromeric heterochromatin and binding to DNA indicating a haploinsufficient mechanism of action for IKZF1L132P Furthermore, Ikzf1+/L132P mice have late onset defective Ig production, similar to what is observed in common variable immunodeficiency patients. RNA sequencing revealed a total loss of Hsf1 expression in follicular B cells, suggesting a possible functional link for the humoral immune response defects observed in Ikzf1L132P/L132P mice.

Increased HSF1 Promotes Infiltration and Metastasis in Cervical Cancer via Enhancing MTDH-VEGF-C Expression.

PurposeTo explore the molecular mechanism of promoting cervical cancer by HSF1 in vivo and in vitro.MethodsThe expression of HSF1 in 110 paraffin-embedded cervical cancer sections of different grades was examined via immunohistochemistry analyses. Expression of HSF1 downstream targets Metadherin (MTDH), VEGF-C and CD31 were studied using immunohistochemistry analyses. HSF1 transcriptional activity in the MTDH promoter region was detected by EMSA, CHIP and luciferase. Cell proliferation and clonality were detected by MTT and clonal formation assay. Cell migration and invasion ability were investigated by scratch analysis and transwell assay. HSF1-mediated tumorigenesis in vivo was examined in xenograft models.ResultsHSF1 expression of cervical cancer cell line was increased compared to normal human cervical tissues. HSF1 enhanced the expression of MTDH, VEGF-C and CD31. HSF1 can combine with MTDH promoter to promote the expression of MTDH. HSF1 enhanced HeLa cell proliferation and clone formation. Furthermore, HSF1 increased HeLa cells migration and invasion in vitro. In the transplanted tumor model, HSF1 inhibited tumor growth in vivo after interference, and reduced the expression of MTDH, VEGF-C and CD31.DiscussionHSF1 can promote the proliferation, metastasis and invasion of cervical cancer.

Hif-1α/Hsf1/Hsp70 signaling pathway regulates redox homeostasis and apoptosis in large yellow croaker ( Larimichthys crocea) under environmental hypoxia.

Oxygen is an essential molecule for animal respiration, growth, and survival. Unlike in terrestrial environments, contamination and climate change have led to the frequent occurrence of hypoxia in aquatic environments, thus impacting aquatic animal survival. However, the adaptative mechanisms underlying fish responses to environmental hypoxia remain largely unknown. Here, we used large yellow croaker (Larimichthys crocea) and large yellow croaker fry (LYCF) cells to investigate the roles of the Hif-1α/Hsf1/Hsp70 signaling pathway in the regulation of cellular redox homeostasis, and apoptosis. We confirmed that hypoxia induced the expression of Hif-1α, Hsf1, and Hsp70 in vivo and in vitro. Genetic Hsp70 knockdown/overexpression indicated that Hsp70 was required for maintaining redox homeostasis and resisting oxidative stress in LYCF cells under hypoxic stress. Hsp70 inhibited caspase-dependent intrinsic apoptosis by maintaining normal mitochondrial membrane potential, enhancing Bcl-2 mRNA and protein expression, inhibiting Bax and caspase3 mRNA expression, and suppressing caspase-3 and caspase-9 activation. Hsp70 suppressed caspase-independent intrinsic apoptosis by inhibiting nuclear translocation of apoptosis-inducing factor (AIF) and disturbed extrinsic apoptosis by inactivating caspase-8. Genetic knockdown/overexpression of Hif-1α and dual-luciferase reporter assay indicated that Hif-1α activated the Hsf1 DNA promoter and enhanced Hsf1 mRNA transcription. Hsf1 enhanced Hsp70 mRNA transcription in a similar manner. In summary, the Hif-1α/Hsf1/Hsp70 signaling pathway plays an important role in regulating redox homeostasis and anti-apoptosis in L. crocea under hypoxic stress.

Trigonelline Extends the Lifespan of C. Elegans and Delays the Progression of Age-Related Diseases by Activating AMPK, DAF-16, and HSF-1.

Trigonelline is the main alkaloid with bioactivity presented in fenugreek, which was used in traditional medicine in Asian countries for centuries. It is reported that trigonelline has anti-inflammatory, anti-oxidant, and anti-pathogenic effects. We are wondering whether trigonelline have anti-aging effect. We found that 50 μM of trigonelline had the best anti-aging activity and could prolong the lifespan of Caenorhabditis elegans (C. elegans) by about 17.9%. Trigonelline can enhance the oxidative, heat, and pathogenic stress resistance of C. elegans. Trigonelline could also delay the development of neurodegenerative diseases, such as AD, PD, and HD, in models of C. elegans. Trigonelline could not prolong the lifespan of long-lived worms with loss-of-function mutations in genes regulating energy and nutrition, such as clk-1, isp-1, eat-2, and rsks-1. Trigonelline requires daf-16, hsf-1, and aak-2 to extend the lifespan of C. elegans. Trigonelline can also up-regulate the expression of daf-16 and hsf-1 targeted downstream genes, such as sod-3, gst-4, hsp-16.1, and hsp-12.6. Our results can be the basis for developing trigonelline-rich products with health benefits, as well as for further research on the pharmacological usage of trigonelline.

CHARACTERIZATION OF PUTATIVE HEAT SHOCK TRANSCRIPTION FACTOR (Hsf2) GENE INVOLVED IN REGULATING THE EXPRESSION OF HSP90 IN WHEAT UNDER TERMINAL HEAT STRESS

Wheat, being staple food grain crop, is highly sensitive to heat stress. The effect is more evident under present threat of global climate change. Very limited information about heat-responsive transcription factor is known in wheat. A putative Heat Shock Factor (HSF) was cloned from wheat under HS. The gene was titled as Hsf2 (acc No. KP063542). The nucleotide sequence was found to be 1551 bp long with open reading frame (ORF) of 1125 bp (5’UTR of 213bp and 3’UTR of 213bp). The amino acid sequence showed the presence of HSF_ DNA binding domain having high degree of similarity (homology) with other HSF coding gene of related species (Aegilops tauschii with 95% homology and 100% homology with Chinese spring cultivar of Triticum aestivum). The protein was observed to be localized in the nucleus. Phosphorylation study showed the presence of phosphorylated threonine at seventeen sites in amino acid sequence. Expression analysis of Hsf2 showed maximum relative fold expression (2.04fold) in the leaves of HD2967 under HS, as compared to control. The transcript abundance was observed maximum in thermotolerant cv., as compared to thermo susceptible. Current study established positive correlation between the expression of Hsf2 and HSP90 under HS. Hsf2, being regulator of HSPs can be used as potential molecular marker for screening wheat germplasm for thermo tolerance. It will pave the way for the development of ‘climate-smart’ wheat crop.

Effects of LRP1B Regulated by HSF1 on Lipid Metabolism in Hepatocellular Carcinoma.

BackgroundTo date, aberrated lipid metabolism has been recognized as an important feature of hepatocellular carcinoma (HCC); however, it remains poorly defined. As a large member of the low-density lipoprotein receptor family, LRP1B plays a pivotal role in maintaining lipid homeostasis. Here we investigated the expression feature of LRP1B in HCC and elucidated its effects on lipid metabolism of HCC cells.Materials and MethodsLRP1B expression in HCC cells and tumor tissues was respectively examined by quantitative PCR, Western blotting and immunohistochemistry. Crispr-cas9 RNA inference and CRISPRa transcription activation system were used to downregulate and upregulate LRP1B expression, respectively. Oil red O staining, DiD staining combined with flow cytometry and transmission electron microscopy were used to evaluate the lipid content in HCC cells. Overall survival (OS) and time to recurrence (TTR) were calculated; meanwhile, Kaplan–Meier and the Cox proportional hazards model were used to assess the prognosis of HCC patients.ResultsIn contrast to inactivation expression in a majority of cancers, LRP1B showed predominantly strong expression in HCC. LRP1B knockdown induced the decrease of intracellular lipid content, downregulated expressions of lipid synthesis-related enzymes and upregulated expressions of β-oxidation-related enzymes as well as activated the AMPK signaling. Moreover, HSF1 directly regulated the transcription of LRP1B and was involved in LRP1B-mediated lipid metabolism in HCC; meanwhile, the combination of LRP1B knockdown and HSF1 inhibition suppressed synergistically the proliferation of HCC cells. In addition, simultaneous expression of HSF1 and LRP1B was an independent prognostic factor for HCC patients.ConclusionAltogether, the study reveals a novel unique role of LRP1B in HCC by serving as a mediator in lipid metabolism, which provides an insight for making explorable therapeutic strategies for HCC.

Elucidating the Neuroprotective Role of Formulated Camel α-Lactalbumin-Oleic Acid Complex by Curating the SIRT1 Pathway in Parkinson's Disease Model.

Parkinson's Disease (PD) is characterized by increased oxidative stress and decreased level of dopamine. At present, the therapeutic interventions of PD are associated with undesirable adverse effects. To overcome these side effects, a new candidate bioinspired molecule is needed for the management of PD. Camel α-lactalbumin (α-LA) is the most abundant protein in camel's milk and has a potential to act as a nutraceutical supplement for neurological functions. Oleic acid, a monounsaturated fatty acid, has been widely associated with a reduced risk of PD. The present study aimed to formulate the camel α-LA and oleic acid (CLOA) complex under specific conditions and to evaluate its efficacy as a neuroprotective in rotenone induced PC12 cell model of PD. Our results demonstrated that removal of Ca++ ions from camel α-LA by EDTA enhances its binding efficiency with oleic acid, and the complex was characterized by UV-CD, ANS fluorescence spectroscopy, and NMR spectroscopy. Moreover, CLOA complex treatment reduced the oxidative stress and increased the cell viability by enhancing the level of dopamine and the expression of SIRT1, FOXO3a, HIF-1α, and HSF-1. We also validated the neuroprotective role of the complex by incubating the cells with CLOA complex prior to rotenone treatment. We inferred from the outcome of the results that the individual entity, i.e., α-LA or OA, is not as effective as the complex. Taken together, our study indicates that CLOA complex might be a potential candidate for the development of future therapeutic drugs for PD.

Effect of long-term treatment with a mixture of pyrethroids on the expression of genes that govern male germ cell production in rats.

Humans are exposed to pyrethroid-based pesticides through agricultural produce. In this study, male Wistar rats were orally treated for 9 to 12 months with a mixture of pyrethroids that is equivalent to one-fifth (high dose; HD) or one-twenty fifth (low dose; LD) of the amount of pyrethroids present in the cereals and rice consumed by an average Indian. In rats treated for 9 months, the spermatogenesis-associated genes Abp, Ar, Cd9, Dax1, Dazap1, Ddx3y, Gdnf, Gfra1, Grth, Inhb, Ovol1, P1, Plzf, Pygo2, Scf, Tgfb1, Tp1, Tp2, and Vim1 were downregulated in both LD and HD groups. In rats treated for 12 months Gdnf, Hsf2, Inhb, Tgfb1, Thy1, and Ybx2 expression was downregulated in both LD and HD groups. Steroidogenesis-associated genes 17-β-Hsd, Gata4, Hmgcr, Hmgcs1, Pde4b, and Tspo gene expression were reduced in both LD- and HD-treated groups treated for 9 months. In 12-month-treated rats, Creb1 expression decreased in both LD and HD groups. The epigenetic reprogramming-associated genes, Dnmt1, Dnmt3a, Dnmt3b, Hdac10, Hp1bp3, Kat3a Kat3b, Mch2ta, Ncoa7, and Sirt1 were downregulated in both HD and LD groups of 9-months-treated rats. In rats treated for 12 months, Hdac10, Mch2ta, Ncoa7, and Sirt1 messenger RNA levels decreased in both the HD and LD groups. Thus, we demonstrate that long-term exposure to a mixture of pyrethroids caused aberrations in the transcriptome of factors involved in sperm production and development.

Quercetin enhances motility in aged and heat-stressed Caenorhabditis elegans nematodes by modulating both HSF-1 activity, and insulin-like and p38-MAPK signalling.

Quercetin is a yellow pigment that is found in many common dietary plants, and that protects against oxidative stress, inflammation, and arteriosclerosis. It has also been suggested to prolong the lifespan of, and enhance heat-stress tolerance in nematodes; thus, the present study investigated its effects on both the nematode life- and health span by assessing its capacity to promote nematode motility after aging and/or heat stress, as well as the mechanisms underlying these effects. The results of the conducted analyses showed that quercetin feeding prolonged lifespan, suppressed age-related motility retardation, improved motility recovery after heat stress, and decreased the production of both intercellular and mitochondrial reactive oxygen species in the analysed Caenorhabditis elegans strains, likely by modulating the insulin-like signalling (ILS) pathway and p38-mitogen-activated protein kinase (MAPK) pathway. In particular, the transcription factors DAF-16 and SKN-1 were found to mediate the observed quercetin-induced effects, consistent with their previously demonstrated roles as regulators of aging. Furthermore, we demonstrated, for the first time, that quercetin induced heat-stress tolerance in C. elegans by modulating HSF-1 expression and/or activity. Thus, the present study provides valuable insights into the mechanisms by which quercetin inhibit aging and enhance heat-stress tolerance via ILS and MAPK pathway in C. elegans.

Caenorhabditis elegans AF4/FMR2 Family Homolog affl-2 Regulates Heat-Shock-Induced Gene Expression.

To mitigate the deleterious effects of temperature increases on cellular organization and proteotoxicity, organisms have developed mechanisms to respond to heat stress. In eukaryotes, HSF1 is the master regulator of the heat shock transcriptional response, but the heat shock response pathway is not yet fully understood. From a forward genetic screen for suppressors of heat-shock-induced gene expression in Caenorhabditis elegans, we found a new allele of hsf-1 that alters its DNA-binding domain, and we found three additional alleles of sup-45, a previously molecularly uncharacterized genetic locus. We identified sup-45 as one of the two hitherto unknown C.elegans orthologs of the human AF4/FMR2 family proteins, which are involved in regulation of transcriptional elongation rate. We thus renamed sup-45 as affl-2 (AF4/FMR2-Like). Through RNA-seq, we demonstrated that affl-2 mutants are deficient in heat-shock-induced transcription. Additionally, affl-2 mutants have herniated intestines, while worms lacking its sole paralog (affl-1) appear wild type. AFFL-2 is a broadly expressed nuclear protein, and nuclear localization of AFFL-2 is necessary for its role in heat shock response. affl-2 and its paralog are not essential for proper HSF-1 expression and localization after heat shock, which suggests that affl-2 may function downstream of, or parallel to, hsf-1 Our characterization of affl-2 provides insights into the regulation of heat-shock-induced gene expression to protect against heat stress.

Hemiterpene compound, 3,3-dimethylallyl alcohol promotes longevity and neuroprotection in Caenorhabditis elegans.

Terpenes and their derivatives have been used conventionally as potential dietary supplements to boost the nutritional value of endless food products. Several plant-based complex terpenoid and their derivatives have been reported for a wide range of medicinal and nutritional properties. However, their simple counterparts, whose production is relatively easy, sustainable, and economic from food-grade microbial sources, have not been studied yet for any such biological activities. The present study aimed to investigate the longevity-promoting property and neuromodulatory effects of 3,3-dimethylallyl alcohol (Prenol), one of the simplest forms of terpenoid and a constituent of fruit aroma, in the animal model Caenorhabditis elegans. Prenol supplementation (0.25mM) augmented the lifespan of wild-type nematodes by 22.8% over the non-treated worms. Moreover, a suspended amyloid-β induced paralysis and reduced α-synuclein aggregation were observed in Prenol-treated worms. The lifespan extending properties of Prenol were correlated with ameliorated physiological parameters and increased stress (heat and oxidative) tolerance in C. elegans. In silico and gene-specific mutant studies showed that pro-longevity transcription factors DAF-16, HSF-1, and SKN-1 were involved in the improved lifespan and health-span of Prenol-treated worms. Transgenic green fluorescent protein-reporter gene expression analysis and relative mRNA quantification (using real-time PCR) demonstrated an increase in the expression of DAF-16, HSF-1, and SKN-1 transcription factors and their downstream target genes in Prenol-treated worms. Together, the findings suggest that small molecules, like Prenol, could be explored as a potential alternate to develop therapeutics against aging and age-related ailments.

Effect of heat stress during the early and late dry period on mammary gland development of Holstein dairy cattle

Dry period heat stress impairs subsequent milk yield. Our objective was to evaluate the effect of heat stress or cooling during the early and late dry period on mammary gland gene expression and microstructure. Cows were dried off ∼45 d before expected parturition and randomly assigned to 1 of 2 treatments: heat stress (HT, n = 39) or cooling (CL, n = 39) during the first 21 d of the dry period. On d 22, cows were switched or remained on HT and CL and this yielded 4 treatments: heat stress during the entire dry period (HTHT, n = 18); cooling during the entire dry period (CLCL, n = 20); HT for the first 21 d dry, then CL until calving (HTCL, n = 21); or CL for the first 21 d dry, then HT until calving (CLHT, n = 19). Data were analyzed in 2 periods: first 21 d dry (early dry period) and from 22 d until calving (late dry period) and analyzed using PROC MIXED or GLM in SAS (SAS Institute Inc., Cary, NC). Mammary biopsies (5-8 cows/treatment) were collected at -3, 3, 7, 14, and 25 d relative to dry-off to evaluate mammary gland gene expression and histology [i.e., cellular apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labeling) and proliferation (Ki67)]. Mammary alveoli number and connective tissue were visualized by hematoxylin and eosin and Mason's trichrome staining, respectively. During the early dry period, CL upregulated expression of CASP3, IGF1R, HSP90, HSF1, BECN1, ATG3, ATG5, and PRLR-LF relative to HT. However, in the late dry period, CLHT treatment upregulated expression of CASP3, CASP8, HSP70, HSP90, PRLR-LF, STAT5, CSN2, and ATG3 relative to CLCL. During the early dry period, cows exposed to HT had reduced mammary and stroma cell apoptosis and proliferation relative to CL. In addition to these findings, cows exposed to HT had lower connective tissue 3 d after dry-off relative to CL. However, in the late dry period, HTHT cows had higher connective tissue relative to CLCL. Also, in the early dry period, cows exposed to HT had greater alveoli number relative to CL, and HT decreased expression of genes related to autophagy and apoptosis in the early dry period, consistent with a delay in involution with HT. Thus, cows exposed to HT have extended involution with delayed apoptosis and autophagy signaling. Also, HT compromises mammary gland cell proliferation and leads to higher connective tissue later in the dry period. These results provide evidence that heat stress impairs overall mammary gland turnover during the dry period, which then affects secretory activity and productivity in the next lactation.

Cloning and identification of grass carp transcription factor HSF1 and its characterization involving the production of fish HSP70.

In mammals, heat shock transcription factor 1 (HSF1) is well documented as the critical transcript factor to regulate heat shock protein 70 (HSP70) expression under different stresses, such as heat shock or bacterial infection. In fish, Hsf1 responses to physiological and environmental stresses and regulates Hsp70 expression under thermal exposure. However, the functional role of Hsf1 in Hsp70 production is still elusive under bacterial infection. In the present study, a coding sequence of grass carp hsf1 (gchsf1) gene was cloned and identified. Using Ctenopharyngodon idellus kidney (CIK) cells as the model, we found that lipopolysaccharide (LPS) exerted stimulatory effects on the expression of grass carp hsp70 (gchsp70) and hsf1, implying possible relationship of Hsp70 and Hsf1 under immune stimulation in fish. To validate the hypothesis, overexpression of gcHsf1 was performed in CIK cells, and the effects of overexpressing gcHsf1 on the expression of gcHsp70 in the absence or presence of LPS were examined. Results showed that LPS significantly upregulated the transcription and protein synthesis of gcHsp70, and these stimulatory effects were further amplified when overexpression of gcHsf1 was performed. Furthermore, luciferase reporter assays in CIK cells revealed that both overexpression of Hsf1 and LPS upregulated gchsp70 transcription, and their combined treatment further enhanced the gchsp70 promoter activity. Moreover, the regions responsive to these treatments were mapped to the promoter of gchsp70. Besides transcriptional level and cellular protein contents, gcHsp70 secretion was measured by competitive ELISA, uncovering that gcHsf1 enhanced the release of gcHsp70 induced by LPS in the same cells. These data not only demonstrated the enhancement of Hsf1 in Hsp70 production but also initially revealed the involvement of Hsf1-Hsp70 axis in mediating inflammatory response in fish.

The proteostasis guardian HSF1 directs the transcription of its paralog and interactor HSF2 during proteasome dysfunction.

Protein homeostasis is essential for life in eukaryotes. Organisms respond to proteotoxic stress by activating heat shock transcription factors (HSFs), which play important roles in cytoprotection, longevity and development. Of six human HSFs, HSF1 acts as a proteostasis guardian regulating stress-induced transcriptional responses, whereas HSF2 has a critical role in development, in particular of brain and reproductive organs. Unlike HSF1, that is a stable protein constitutively expressed, HSF2 is a labile protein and its expression varies in different tissues; however, the mechanisms regulating HSF2 expression remain poorly understood. Herein we demonstrate that the proteasome inhibitor anticancer drug bortezomib (Velcade), at clinically relevant concentrations, triggers de novo HSF2 mRNA transcription in different types of cancers via HSF1 activation. Similar results were obtained with next-generation proteasome inhibitors ixazomib and carfilzomib, indicating that induction of HSF2 expression is a general response to proteasome dysfunction. HSF2-promoter analysis, electrophoretic mobility shift assays, and chromatin immunoprecipitation studies unexpectedly revealed that HSF1 is recruited to a heat shock element located at 1.397bp upstream from the transcription start site in the HSF2-promoter. More importantly, we found that HSF1 is critical for HSF2 gene transcription during proteasome dysfunction, representing an interesting example of transcription factor involved in controlling the expression of members of the same family. Moreover, bortezomib-induced HSF2 was found to localize in the nucleus, interact with HSF1, and participate in bortezomib-mediated control of cancer cell migration. The results shed light on HSF2-expression regulation, revealing a novel level of HSF1/HSF2 interplay that may lead to advances in pharmacological modulation of these fundamental transcription factors.

721 Abnormal autophagosome formation increased melanocyte sensitivity to H2O2-induced oxidative stress in vitiligo

Autophagy is a controlled self-digestion process which can protect cells against oxidative damage. Dysregulated autophagy has been demonstrated to increase melanocyte sensitivity to oxidative stress in vitiligo. However, the exact mechanism is still not clear. This study aimed to determine the implications of autophagy for melanocyte survival in response to oxidative stress. Our results demonstrated that the autophagic flux in PIG1 exposure to H2O2 was significantly enhanced compared with that in PIG3V, which were accompanied by high level of ROS accumulation, membrane potential changes, and increased apoptosis. It indicates that vitiligo melanocytes exhibited hypersensitivity to H2O2-induced oxidative injury due to dysregulated autophagy. To further explore the mechanism, we performed RNA sequencing to compare the RNA expression in PIG1 and PIG3V cells exposed to H2O2, the bioinformatic analysis indicate that autophagosome formation was impaired in vitiligo melanocytes, our in vitro study also showed that inhibition of autolysosome degradation can not lead to autophagosome accumulation in vitiligo melanocytes, confirming that the impairment of autophagosome formation is responsible for the defects of autophagy in vitiligo melanocytes, the further study also showed that overexpression of HSF1, the main transcription factor for Atg5 and Atg12, could reduce H2O2-induced oxidative damage of vitiligo melanocytes. Our data demonstrated that owing to the deficiency of HSF1 expression, the autophagosome formation was blocked, which resulted in autophagy impairment and further increase the sensitivity of vitiligo melanocytes to oxidative stress. Our results indicating that targeting autophagy may be a potential therapy option.

Molecular Mechanism of HSF1-Upregulated ALDH2 by PKC in Ameliorating Pressure Overload-Induced Heart Failure in Mice.

Evidences abound that HSF1 and ALDH2 are of cardioprotective effect, yet there is still no report on whether HSF1 can regulate ALDH2 to delay the occurrence of heart failure. We first established the pressure overload-induced heart failure model of mice by transverse aortic constriction (TAC) and discovered that, in the forming period of heart failure, changes of HSF1 and ALDH2 expression recorded the consistent trend. When HSF1 was upregulated/downregulated to delay/promote the occurrence of heart failure, PKC and ALDH2 also showed increased/decreased expression. And when ALDH2 was upregulated/downregulated, the role of HSF1 in delaying the occurrence of heart failure strengthened/weakened. Next, we used mechanical stretch to establish a pressure-stimulated myocardial hypertrophy model and discovered an increased expression of both HSF1 and ALDH2. When HSF1 was upregulated/downregulated to increase/decrease the expression of myocardial hypertrophy gene beta-MHC, PKC and ALDH2 recorded an increased/decreased expression. When an inhibitor was used to downregulate the expression of PKC in cardiomyocytes, we found that the role of HSF1 in upregulating ALDH2 beta-MHC weakened. These findings suggest that HSF1 can upregulate the expression of ALDH2 via PKC to promote pressure-stimulated myocardial compensatory hypertrophy, which is an important molecular pathway for HSF1 to ameliorate heart failure.

Transcription factor YY1 inhibits the expression of THY1 to promote interstitial pulmonary fibrosis by activating the HSF1/miR-214 axis

Interstitial pulmonary fibrosis (IPF) is a progressive disease of diverse etiology manifesting with proliferation of lung fibroblasts and accumulation of extracellular matrix deposition in pulmonary interstitium. Recent studies show aberrant expression of mRNAs and microRNAs (miRNAs) in human embryonic pulmonary fibroblasts (HEPFs). In this study, we investigated effects of the YY1/HSF1/miR-214/THY1 axis on the functions of HEPFs and IPF. Loss- and gain-of-function tests were conducted to identify roles of YY1, HSF1, miR-214, and THY1 in IPF. As determined by RT-qPCR or western blot assay, silencing YY1 down-regulated HSF1 expression and attenuated the expression of pro-proliferative and fibrosis markers in HEPFs. Meanwhile, viability of HEPFs was impeded by YY1 knockdown. The binding relationship between miR-214 and THY1 was verified using dual-luciferase reporter assay. In HEPFs, down-regulation of HSF1 reduced miR-214 expression to repress proliferation and fibrogenic transformation of HEPFs, while inhibition of miR-214 expression could restrain the fibrogenic transformation property of HEPFs by up-regulating THY1. Subsequently, IPF model in mice was induced by bleomycin treatment. These animal experiments validated the protective effects of YY1 knockdown against IPF-induced lung pathological manifestations, which could be reversed by THY1 knockdown. Our study demonstrates the important involvement of YY1/HSF1/miR-214/THY1 axis in the development of IPF.

Decreased expression of FBXW7 by ERK1/2 activation in drug-resistant cancer cells confers transcriptional activation of MDR1 by suppression of ubiquitin degradation of HSF1

The acquisition of MDR1-mediated chemoresistance poses a major obstacle to the success of conventional chemotherapeutic agents. HSF1 is also involved in chemoresistance, and several studies have demonstrated the relationship between HSF1 and MDR1 but without any consistent results. Paclitaxel- and doxorubicin-resistant cancer cells showed higher expression of MDR1 and HSF1. Depletion of HSF1 decreased mdr1 expression at mRNA level, and HSF1 directly interacted with the promoter site of mdr1, suggesting its role as a transcriptional regulator of MDR1. Phosphorylation of Ser303/307, which was involved in protein stability of HSF1 by FBXW7-mediated degradation, was found to be important for transcriptional activation of mdr1. Drug-resistant cells showed decreased expression of FBXW7, which was mediated by the activation of ERK1/2, thus indicating that over-activation of ERK1/2 in drug-resistant cells decreased FBXW7 protein stability, which finally inhibited protein degradation of pHSF1 at Ser303/307. There was a positive correlation between immunofluorescence data of pHSF1 at Ser303/307 and MDR1 in carcinogen-induced rat mammary tumors and human lung cancers. These findings identified the post-translational mechanisms of HSF1 transcription in MDR1 regulation of drug resistance development.