Heat Shock Transcription Factor 1 Protein Research Articles

Heat stress–associated changes in the intestinal barrier, inflammatory signals, and microbiome communities in dairy calves

Recent studies indicate that heat stress pathophysiology is associated with intestinal barrier dysfunction, local and systemic inflammation, and gut dysbiosis. However, inconclusive results and a poor description of tissue-specific changes must be addressed to identify potential intervention targets against heat stress illness in growing calves. Therefore, the objective of this study was to evaluate components of the intestinal barrier, pro- and anti-inflammatory signals, and microbiota community composition in Holstein bull calves exposed to heat stress. Animals (mean age = 12 wk old; mean body weight = 122 kg) penned individually in temperature-controlled rooms were assigned to (1) thermoneutral conditions (constant room temperature at 19.5°C) and restricted offer of feed (TNR, n = 8), or (2) heat stress conditions (cycles of room temperatures ranging from 20 to 37.8°C) along with ad libitum offer of feed (HS, n = 8) for 7 d. Upon treatment completion, sections of the jejunum, ileum, and colon were collected and snap-frozen immediately to evaluate gene and protein expression, cytokine concentrations, and myeloperoxidase activity. Digesta aliquots of the ileum, colon, and rectum were collected to assess bacterial communities. Plasma was harvested on d 2, 5, and 7 to determine cytokine concentrations. Overall, results showed a section-specific effect of HS on intestinal integrity. Jejunal mRNA expression of TJP1 was decreased by 70.9% in HS relative to TNR calves. In agreement, jejunal expression of heat shock transcription factor-1 protein, a known tight junction protein expression regulator, decreased by 48% in HS calves. Jejunal analyses showed that HS decreased concentrations of IL-1α by 36.6% and tended to decrease the concentration of IL-17A. Conversely, HS elicited a 3.5-fold increase in jejunal concentration of anti-inflammatory IL-36 receptor antagonist. Plasma analysis of pro-inflammatory cytokines showed that IL-6 decreased by 51% in HS relative to TNR calves. Heat stress alteration of the large intestine bacterial communities was characterized by increased genus Butyrivibrio_3, a known butyrate-producing organism, and changes in bacteria metabolism of energy and AA. A strong positive correlation between the rectal temperature and pro-inflammatory Eggerthii spp. was detected in HS calves. In conclusion, this work indicates that HS impairs the intestinal barrier function of jejunum. The pro- and anti-inflammatory signal changes may be part of a broader response to restore intestinal homeostasis in jejunum. The changes in large intestine bacterial communities favoring butyrate-producing organisms (e.g., Butyrivibrio spp.) may be part of a successful response to maintain the integrity of the colonic mucosa of HS calves. The alteration of intestinal homeostasis should be the target for heat stress therapies to restore biological functions, and, thus highlights the relevance of this work.

HSF1 promotes CD69+ Treg differentiation to inhibit colitis progression.

Regulatory T cells (Tregs) are critical for generating and maintaining peripheral tolerance. Treg-based immunotherapy is valuable for the clinical management of diseases resulting from dysregulation of immune tolerance. However, the lack of potency is a potential limitation of Treg therapy. In addition, CD69 positive-Treg (CD69+ Treg) represent a newly identified subset of Tregs with potent immune suppressive capability. Methods: Foxp3 YFP-Cre CD69 fl/fl and CD4 Cre CD69 fl/fl mice were generated to determine the relevance of CD69 to Treg. Chromatin Immunoprecipitation Assay (ChIP) and luciferase Assay were performed to detect the regulation of CD69 transcription by heat shock transcription factor 1(HSF1). Gene expression was measured by western blotting and qRT-PCR. The differentiation of naive T cells to CD69+Foxp3+ iTregs was determined by flow cytometry. The immunosuppressive ability of Tregs was analyzed by ELISA and flow cytometry. Colon inflammation in mice was reflected by changes in body weight and colon length, the disease activity index (DAI), and H&E staining of colon tissues. Results: Induced Tregs (iTregs) from CD4 Cre CD69 fl/fl mice failed to alleviate colitis. The transcription factor HSF1 interacted with the promoter of the CD69 gene to prompt its transcription during Treg differentiation. Genetic and chemical inhibition of HSF1 impaired CD69+ Treg differentiation and promoted the pathogenesis of colitis in mice. In contrast, HSF1 protein stabilized by inhibiting its proteasomal degradation promoted CD69+ Treg differentiation and alleviated colitis in mice. Moreover, adoptive transfer of iTregs with HSF1 stabilization by proteasome inhibitor (PSI) dramatically prevented the development of colitis in mice and was accompanied by decreased production of pro-inflammatory cytokines and reduced accumulation of pro-inflammatory lymphocytes in colitis tissue, whereas Tregs induced in the absence of PSI were less stable and ineffective in suppressing colitis. Conclusions: HSF1 promotes CD69+ Tregs differentiation by activating the CD69 transcription, which is critical for the immunosuppressive function of Tregs. Stabilization of HSF1 by PSIs results in the efficient generation of Tregs with high potency to treat colitis and probably other autoimmune diseases involving Tregs deficiency.

Protective effect of heat shock transcription factor 1 on acute lung injury in septic rats by regulating NOD-like receptor protein 3 inflammasome activation

To determine whether heat shock transcription factor 1 (HSF1) ameliorates sepsis induced acute lung injury (ALI) by regulating NOD-like receptor protein 3 (NLRP3) inflammasome activity in rat alveolar macrophages (AM). Twenty-four SPF Sprague-Dawley (SD) rats were divided into control group, lipopolysaccharide (LPS) group, overexpression empty vector+LPS group, and overexpression HSF1+LPS group by random number table method, with 6 rats in each group. The rat model of sepsis-induced ALI was reproduced by intraperitoneal injection of LPS (5 mg/kg); the rats in the control group were given the same volume of normal saline. The rats in the overexpression empty vector+LPS group and the overexpression HSF1+LPS group were instilled with 100 μL of overexpressed empty vector adenovirus or overexpressed HSF1 adenovirus through the trachea, respectively; the rats in the control group and LPS group were instilled with an equal volume of normal saline at the same time. At 6 hours after the model was reproduced, carotid blood was collected to determine the oxygenation index (PaO2/FiO2); lung tissue was obtained, and hematoxylin-eosin (HE) staining was performed to observe the pathological changes of lung tissue under a light microscope. Lung tissue wet/dry ratio (W/D) was determined. Immunohistochemistry was used to detect the positive expression of macrophage-specific marker antibody CD68. Western blotting was used to detect the protein expressions of HSF1 and NLRP3. Bronchoalveolar lavage fluid (BALF) was collected, and the levels of interleukins (IL-1β, IL-18) were detected by enzyme-linked immunosorbent assay (ELISA). In addition, BALF of normal rats was collected, and primary AM was isolated, cultured and divided into four groups. The AM in the blank control group was cultured normally without any treatment; the LPS group was treated with 1 mg/L LPS for 24 hours to reproduced the LPS stimulation model; AM in the overexpression empty vector+LPS group and the overexpression HSF1+LPS group were transfected with empty vector plasmid or overexpressed HSF1 plasmid, respectively, for 48 hours, and then the AM was treated with 1 mg/L LPS. The cell viability was detected by cell counting kit-8 (CCK-8). The protein expressions of HSF1 and NLRP3 in AM were detected by Western blotting. The levels of IL-1β and IL-18 in AM culture medium were determined by ELISA. Compared with the control group, the rat lung structure in the LPS group was severely injured, the alveolar cavity and pulmonary interstitium were congested and edema, the alveolar walls were significantly thickened and ruptured, accompanied by a large number of inflammatory cells infiltration. The lung W/D ratio increased, the infiltration degree of macrophages increased, PaO2/FiO2 decreased, HSF1 protein expression decreased and NLRP3 protein expression increased in lung tissue and AM, and IL-1β and IL-18 levels in BALF and AM culture medium increased. Compared with the LPS group, the degree of lung injury in the overexpression HSF1+LPS group was significantly improved, the lung W/D ratio was significantly reduced (4.76±0.16 vs. 6.93±0.33, P < 0.05), the macrophage infiltration was reduced, PaO2/FiO2 increased significantly [mmHg (1 mmHg ≈ 0.133 kPa): 397.62±19.46 vs. 280.12±37.42, P < 0.05], HSF1 protein expression was significantly up-regulated in lung tissue and AM (HSF1/GAPDH: 0.90±0.04 vs. 0.61±0.04 in lung tissue, 1.10±0.10 vs. 0.57±0.08 in AM, both P < 0.05), NLRP3 protein expression was significantly down-regulated (NLRP3/GAPDH: 0.75±0.14 vs. 1.05±0.11 in lung tissue, 0.81±0.09 vs. 1.14±0.17 in AM, both P < 0.05), and the contents of IL-1β and IL-18 in BALF and AM medium were significantly decreased [IL-1β (ng/L): 7.82±0.45 vs. 14.09±0.58 in BALF, 11.11±0.46 vs. 16.66±0.96 in AM; IL-18 (ng/L): 50.44±3.30 vs. 66.31±5.67 in BALF, 43.95±0.88 vs. 73.52±1.23 in AM, all P < 0.05]. There was no significant difference in the detection indicators between the overexpression empty vector+LPS group and the LPS group. HSF1 attenuates LPS-induced ALI in rats, and the mechanism may be related to the inhibition of NLRP3 inflammasome activation in AM.

Ovarian tumorB1-mediated heat shock transcription factor 1 deubiquitination is critical for glycolysis and development of endometriosis

Endometriosis is a common chronic condition characterized by abnormal growth of the endometrium outside the uterus. Heat shock transcription factor 1 (HSF1) is a significant regulator of the proteotoxic stress response and plays an essential role in developing endometriosis. However, the mechanisms regulating HSF1 protein stability in endometriosis remain unclear. Here, we demonstrate that OTUB1 interacts with HSF1 and promotes HSF1 protein stability through deubiquitination. In addition, OTUB1 enhances glycolysis and epithelial-mesenchymal transition of endometriosis cells, leading to promote proliferation, migration, and invasion of endometriosis cells. The progression of endometriosis is inhibited in an OTUB1-knockout mouse model. In summary, OTUB1 promotes the development of endometriosis by up-regulating HSF1. OTUB1/HSF1 axis may become a new therapeutic target for endometriosis.

Comparative interactomes of HSF1 in stress and disease reveal a role for CTCF in HSF1-mediated gene regulation

Heat shock transcription factor 1 (HSF1) orchestrates cellular stress protection by activating or repressing gene transcription in response to protein misfolding, oncogenic cell proliferation, and other environmental stresses. HSF1 is tightly regulated via intramolecular repressive interactions, post-translational modifications, and protein-protein interactions. How these HSF1 regulatory protein interactions are altered in response to acute and chronic stress is largely unknown. To elucidate the profile of HSF1 protein interactions under normal growth and chronic and acutely stressful conditions, quantitative proteomics studies identified interacting proteins in the response to heat shock or in the presence of a poly-glutamine aggregation protein cell-based model of Huntington's disease. These studies identified distinct protein interaction partners of HSF1 as well as changes in the magnitude of shared interactions as a function of each stressful condition. Several novel HSF1-interacting proteins were identified that encompass a wide variety of cellular functions, including roles in DNA repair, mRNA processing, and regulation of RNA polymerase II. One HSF1 partner, CTCF, interacted with HSF1 in a stress-inducible manner and functions in repression of specific HSF1 target genes. Understanding how HSF1 regulates gene repression is a crucial question, given the dysregulation of HSF1 target genes in both cancer and neurodegeneration. These studies expand our understanding of HSF1-mediated gene repression and provide key insights into HSF1 regulation via protein-protein interactions.

Phosphorylation of HSF1 by PIM2 Induces PD-L1 Expression and Promotes Tumor Growth in Breast Cancer.

Heat shock transcription factor 1 (HSF1) is the master regulator of the proteotoxic stress response, which plays a key role in breast cancer tumorigenesis. However, the mechanisms underlying regulation of HSF1 protein stability are still unclear. Here, we show that HSF1 protein stability is regulated by PIM2-mediated phosphorylation of HSF1 at Thr120, which disrupts the binding of HSF1 to the E3 ubiquitin ligase FBXW7. In addition, HSF1 Thr120 phosphorylation promoted proteostasis and carboplatin-induced autophagy. Interestingly, HSF1 Thr120 phosphorylation induced HSF1 binding to the PD-L1 promoter and enhanced PD-L1 expression. Furthermore, HSF1 Thr120 phosphorylation promoted breast cancer tumorigenesis in vitro and in vivo. PIM2, pThr120-HSF1, and PD-L1 expression positively correlated with each other in breast cancer tissues. Collectively, these findings identify PIM2-mediated HSF1 phosphorylation at Thr120 as an essential mechanism that regulates breast tumor growth and potential therapeutic target for breast cancer. SIGNIFICANCE: These findings identify heat shock transcription factor 1 as a new substrate for PIM2 kinase and establish its role in breast tumor progression.

MODULATION OF HSF-1 LEVELS BY HIGH CHOLESTEROL AND ITS OXIDIZED PRODUCT 27-HYDROXYCHOLESTEROL

AbstractDietary intake may influence both aging and disease-related genes that increase Alzheimer’s Disease risk. Some diets may improve cognition and people highly adherent to the Mediterranean diet experience less hippocampal atrophy over time thAan those less adherent. Conversely, diets rich in saturated free fatty acids (sFFA) may increase AD risk. The mechanism of fat induced neurodegeneration includes neuroinflammation blood-brain barrier (BBB) disruption, phosphorylated Tau, and proteotoxicity. Thus, diets may either promote or delay aging and AD.We examined the expression of the longevity and stress factor, Heat shock transcription factor 1 (HSF1), in a cholesterol – fed rabbit model of AD. In this model, we found that both the mRNA levels and the protein distribution of HSF1 are significantly decreased in rabbit hippocampi relative to age matched controls. Because 27-OHC levels are elevated by hypercholesterolemia, aging and oxidative stress, we examined in vitro effects of 27-OHC on astrocytes and found dramatic reductions in HSF1 protein levels in the absence of cell death.Collectively, our results suggest that high cholesterol diets and its oxidized metabolites such as 27-OHC negatively impact a key longevity and cell protection factor, HSF1. Dysregulation of HSF1 by cholesterol and/or its oxidative by-products appears to be at the mRNA or transcriptional level, suggesting a heretofore unknown mechanism of HSF1 regulation. Because disturbances in cholesterol metabolism, oxidative stress, and aging are all risk factors for AD, our results provide new information that disruption of HSF1 may be a key link by which these factors lead to AD progression.

Doxorubicin attenuates CHIP-guarded HSF1 nuclear translocation and protein stability to trigger IGF-IIR-dependent cardiomyocyte death.

Doxorubicin (DOX) is one of the most effective antitumor drugs, but its cardiotoxicity has been a major concern for its use in cancer therapy for decades. Although DOX-induced cardiotoxicity has been investigated, the underlying mechanisms responsible for this cardiotoxicity have not been completely elucidated. Here, we found that the insulin-like growth factor receptor II (IGF-IIR) apoptotic signaling pathway was responsible for DOX-induced cardiotoxicity via proteasome-mediated heat shock transcription factor 1 (HSF1) degradation. The carboxyl-terminus of Hsp70 interacting protein (CHIP) mediated HSF1 stability and nuclear translocation through direct interactions via its tetratricopeptide repeat domain to suppress IGF-IIR expression and membrane translocation under physiological conditions. However, DOX attenuated the HSF1 inhibition of IGF-IIR expression by diminishing the CHIP–HSF1 interaction, removing active nuclear HSF1 and triggering HSF1 proteasomal degradation. Overexpression of CHIP redistributed HSF1 into the nucleus, inhibiting IGF-IIR expression and preventing DOX-induced cardiomyocyte apoptosis. Moreover, HSF1A, a small molecular drug that enhances HSF1 activity, stabilized HSF1 expression and minimized DOX-induced cardiac damage in vitro and in vivo. Our results suggest that the cardiotoxic effects of DOX result from the prevention of CHIP-mediated HSF1 nuclear translocation and activation, which leads to an upregulation of the IGF-IIR apoptotic signaling pathway. We believe that the administration of an HSF1 activator or agonist may further protect against the DOX-induced cell death of cardiomyocytes.

NEDD4-mediated HSF1 degradation underlies α-synucleinopathy.

Cellular protein homeostasis is achieved by a delicate network of molecular chaperones and various proteolytic processes such as ubiquitin–proteasome system (UPS) to avoid a build-up of misfolded protein aggregates. The latter is a common denominator of neurodegeneration. Neurons are found to be particularly vulnerable to toxic stress from aggregation-prone proteins such as α-synuclein. Induction of heat-shock proteins (HSPs), such as through activated heat shock transcription factor 1 (HSF1) via Hsp90 inhibition, is being investigated as a therapeutic option for proteinopathic diseases. HSF1 is a master stress-protective transcription factor which activates genes encoding protein chaperones (e.g. iHsp70) and anti-apoptotic proteins. However, whether and how HSF1 is dysregulated during neurodegeneration has not been studied. Here, we discover aberrant HSF1 degradation by aggregated α-synuclein (or α-synuclein-induced proteotoxic stress) in transfected neuroblastoma cells. HSF1 dysregulation via α-synuclein was confirmed by in vivo assessment of mouse and in situ studies of human specimens with α-synucleinopathy. We demonstrate that elevated NEDD4 is implicated as the responsible ubiquitin E3 ligase for HSF1 degradation through UPS. Furthermore, pharmacologically induced SIRT1-mediated deacetylation can attenuate aberrant NEDD4-mediated HSF1 degradation. Indeed, we define the acetylation status of the Lys 80 residue located in the DNA-binding domain of HSF1 as a critical factor in modulating HSF1 protein stability in addition to its previously identified role in the transcriptional activity. Together with the finding that preserving HSF1 can alleviate α-synuclein toxicity, this study strongly suggests that aberrant HSF1 degradation is a key neurodegenerative mechanism underlying α-synucleinopathy.

Development-dependent regulation of molecular chaperones after hypoxia–ischemia

Cellular stress response after hypoxia–Ischemia (HI) may be substantially different between immature and mature brains. To study this phenomenon, postnatal day 7 (P7) and P26 rats were subjected to HI followed by different periods of recovery. Nuclear accumulation of heat-shock transcription factor-1 (HSF1) and expression of molecular chaperone proteins and mRNAs were analyzed by in situ hybridization, Western blotting and confocal microscopy. Nuclear accumulation of HSF1 protein and induction of hsp70 mRNA occurred dramatically in P26 neurons, but minimally in P7 neurons and moderately in microglial cells after HI. Consistently, the level of HSF1 was significantly higher in P26 brain samples, compared with that in P7 brain. Translation of hsp70 mRNA into proteins in P26 mature neurons was seen at 4h and peaked at 24h, when some neurons had already died after HI. Induction of ER glucose-regulated protein-78 (grp78) and mitochondrial hsp60 mRNAs and proteins was moderate and occurred also only in P26 mature brain after HI. These results suggest that the cellular stress response after HI is development-dependent, being pronounced in mature but virtually negligible in neonatal neurons. Therefore, the effectiveness of therapeutic strategies targeting the stress pathway against HI may be significantly different between immature and mature brains. The delayed induction of molecular chaperones in mature brain may be somewhat late for protecting HI neurons from acute HI injury.

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

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

Heat shock transcription factor 1 expression is spatially distributed and selectively down-regulated in the human placenta during labour

Heat shock transcription factor 1 expression is spatially distributed and selectively down-regulated in the human placenta during labour

Deregulation of DNA-dependent protein kinase catalytic subunit contributes to human hepatocarcinogenesis development and has a putative prognostic value

Background:The DNA-repair gene DNA-dependent kinase catalytic subunit (DNA-PKcs) favours or inhibits carcinogenesis, depending on the cancer type. Its role in human hepatocellular carcinoma (HCC) is unknown.Methods:DNA-dependent protein kinase catalytic subuni, H2A histone family member X (H2AFX) and heat shock transcription factor-1 (HSF1) levels were assessed by immunohistochemistry and/or immunoblotting and qRT–PCR in a collection of human HCC. Rates of proliferation, apoptosis, microvessel density and genomic instability were also determined. Heat shock factor-1 cDNA or DNA-PKcs-specific siRNA were used to explore the role of both genes in HCC. Activator protein 1 (AP-1) binding to DNA-PKcs promoter was evaluated by chromatin immunoprecipitation. Kaplan–Meier curves and multivariate Cox model were used to study the impact on clinical outcome.Results:Total and phosphorylated DNA-PKcs and H2AFX were upregulated in HCC. Activated DNA-PKcs positively correlated with HCC proliferation, genomic instability and microvessel density, and negatively with apoptosis and patient's survival. Proliferation decline and massive apoptosis followed DNA-PKcs silencing in HCC cell lines. Total and phosphorylated HSF1 protein, mRNA and activity were upregulated in HCC. Mechanistically, we demonstrated that HSF1 induces DNA-PKcs upregulation through the activation of the MAPK/JNK/AP-1 axis.Conclusion:DNA-dependent protein kinase catalytic subunit transduces HSF1 effects in HCC cells, and might represent a novel target and prognostic factor in human HCC.

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

Heat shock factor‐1 (HSF1) is the master regulator for cytoprotective heat shock protein (Hsp) expression. It is widely thought that HSF1 expression is non‐inducible and thus the key control point of HSP expression is regulation of HSF1's transactivation activity. How HSF1 expression is regulated remains unknown. Herein, we demonstrate that glutamine (Gln) enhanced Hsp expression both in rat colonic epithelium in vivo and in cultured non‐transformed colonic YAMC cells. This was associated with upregulated HSF1's transactivation activity via increased HSF1 trimerization, nuclear localization and DNA binding. Intriguingly, Gln enhanced HSF1 protein and mRNA expression and Hsf1 gene promoter activity. Within the −281/−200 region of the Hsf1 promoter, deletion of the putative CCAAT enhancer binding protein (C/EBP) binding site abolished Gln's HSF1 response. Gln availability strikingly altered the ratio of C/EBPβ inhibitory and active isoforms, i.e., liver‐enriched inhibitory protein (LIP) and liver‐enriched activating protein (LAP). LIP and LAP were further shown to be independent repressor and activator respectively for Hsf1 gene transcription and the relative abundance of these two C/EBPβ isoforms was demonstrated to determine Hsf1 transcription. We show for the first time that Gln not only enhances HSF1's transactivation, but also induces Hsf1 gene transcription in a C/EBPβ‐dependent manner.

Expression and clinical significance of heat shock transcription factor 1 protein in human hepatocellular carcinoma tissues

Objective To investigate the expression and clinical significance of heat shock transcription factor 1 (HSF1) protein in human hepatocellular carcinoma (HCC) tissues,and deduce the probable molecular mechanism of HSF1 in the development and advancement of HCC.Methods Sixty-seven samples of HCC tissue and 21 samples of normal liver tissue were obtained from March 2006 to March 2007 at the Xijing Hospital.The expressions of HSF1 protein and heat shock protein 70 (HSP70) were detected by using immunohistochemistry.The probable molecular mechanism of HSF1 in the development and advancement of HCC was deduced according to the relationship between the expressions of HSF1 protein and HSP70.Positive rates of HSF1 protein in different tissues and the relationship between HSF1 protein expression in the HCC tissues and clinical pathological factors were analyzed by the chi-square test and by calculating Fisher exact probability,respectively.The correlation between the expressions of HSF1 protein and HSP70 in the HCC tissue was analyzed by the Spearman correlation coefficient.The survival curve was drawn by the Kaplan-Meier method,and the survival rate was analyzed by the Log-rank test.Results The positive rates of HSF1 protein expression was 69％ (46/67) in the HCC tissue,which was significantly higher than 29％ (6/21) in the normal liver tissue ( x2 =10.628,P ＜ 0.05 ),The positive rates of HSP70 expression in the HCC tissue was 57％ (38/67),which was significantly higher than 24％ (5/21) in the normal liver tissue ( x2 =6.929,P ＜ 0.05 ).The expression of HSF1 protein in the HCC tissue was positively correlated with that of HSP70 (r=0.319,P ＜0.05).The high expression of HSF1 protein was correlated with the integrity of capsule of HCC,tumor differentiation and TNM stage (x2 =5.935,9.762,5.159,11.267,P＜0.05 ),while the high expression of HSF1 protein was not correlated with the gender,age,levels of hepatitis B surface antigen and alpha fetoprotein,and portal vein tumor thrombus ( x2 =0.822,0.172,2.059,P ＞0.05 ).The survival time was (21.4 ± 1.9 )months for patients with positive HSF1 protein expression and (29.8 ± 2.7 ) months for patients with negative HSF1 protein expression.There was a significant difference in the survival time between patients with positive and negative HSF1 protein expression ( x2 =4.276,P ＜ 0.05 ).Conclusions HSF1 is correlated with the development,advancement,invasion,metastasis and malignant prognosis of HCC.HSF1 takes effects by regulating the expression of HSP70,and it has a good perspective of clinical application for the diagnosis and treatment of HCC. Key words: Liver neoplasms; Heat shock transcription factor 1; Heat shock protein 70; Immunohistochemistry

Abstract 4673A: DAPK-mediated phosphorylation of HSF1 enhances apoptosis level upon TNF in colorectal carcinoma cells

Abstract Objectives: Tumor necrosis factor ≤ (TNF) is an inflammatory cytokine, which is released upon different stimuli, including irradiation. Recently it has been shown, that the Death-associated protein kinase (DAPK) mediates TNF-induced apoptosis in colon cancer cells [1]. Here, we aimed to identify new DAPK binding partners and to characterize the functional role of novel protein interaction complexes during TNF-induced apoptosis in colon cancer cells. Methods/Results: HCT116 colorectal cancer cells were cultured for 6 to 48 hours in either normal or TNF-conditioned medium. For phosphopeptide microarray (PPM) whole cell lysates were incubated on peptide platforms with radioactive-labeled P33. Apoptosis was detected by Annexin V staining and caspase 3 cleavage in Western Blotting. PPM analysis revealed heat shock transcription factor 1 (HSF1) as a new potential substrate of DAPK phosphorylation under TNF-stimulation. DAPK Co-IP, co-immunofluorescence (Co-IF), and mass spectrometry showed that DAPK interacts with HSF1 after TNF-treatment. Phosphorylation on serine residues of HSF1 is necessary for HSF1 nuclear translocation and the initiation of transcription of its target genes. Among them, Ser 230 is part of a consensus phosphorylation motif for DAPK. Maximal level of pHSF1Ser230 was observed at the time point where massive apoptosis was induced. Co-IF microscopy confirmed enrichment of pHSF1Ser230 in the nucleus already after 24 hours of TNF-stimulation. EMSA and Chromatin-IP revealed that pHSF1Ser230 binds to the heat shock response element in the DAPK promoter region and enhances its transcriptional activity. Exogenous over-expression of HSF1 protein led to a significant increase in mRNA DAPK levels and consequently to an enforcement of apoptosis. As expected, DAPK knockdown cells did not show any variation in pHSF1Ser230 level, supporting again that DAPK is an important mediator of the TNF-driven signaling pathway in colorectal cancer cells. The significance of the DAPK/pHSF1Ser230 interaction for response prediction was evaluated by immunohistochemical staining on tissue microarrays of colorectal cancer before and after radiotherapy. Conclusion: Our data show a novel functional interaction between HSF1 and DAPK under TNF-stress and highlight a positive feedback mechanism in DAPK-regulation. These results help to understand cell death pathways in response to radiotherapy. [1].Bajbouj K.et al., Am J Pathol. (2009) 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 4673A. doi:1538-7445.AM2012-4673A

Glutamine activates heat shock transcription factor‐1 (HSF1) gene transcription

We have demonstrated that glutamine (GLN) enhances cytoprotective heat shock protein (HSP) expression. Heat shock transcription factor‐1 (HSF1) is the master regulator for HSP gene expression. We are aimed to explore GLN's effects on HSF1's activity and expression. Non‐stressed or heat‐stressed YAMC colonic epithelial cells were exposed to GLN at 0, 0.5 or 2mM. HSF1 expression, nuclear localization, trimerization and DNA binding were determined. HSF1 promoter‐driven reporter assay and deletion analysis of HSF1 gene promoter were conducted. GLN upregulated HSF1's transactivation activity by increasing its trimerization, nuclear localization and DNA binding. Further, GLN increased HSF1 protein and mRNA expression in both non‐stressed and stressed cells. Deletion analysis of HSF1 promoter shows that the key cis‐ element responsible for GLN response is present between bp −331 to −200. Within this region, deletion of the putative CCAAT enhancer binding protein (C/EBP) binding site resulted in decreased GLN response. In absence of glutamine, HSF1 expression was de‐repressed in C/EBPβ‐silenced cells. Over‐expression of the inhibitory dominant‐negative isoform of C/EBPβ repressed HSF1 expression in presence of adequate glutamine. We for the first time show that GLN not only activates HSF1's transactivation activity, but also activates HSF‐1 gene transcription per se in a C/EBPβ‐dependent manner.

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

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

Activation of heat shock factor 1 by hyperosmotic or hypo-osmotic stress is drastically attenuated in normal human fibroblasts during senescence.

We have previously reported that osmotic stress prominently induces the DNA binding activity of the heat shock transcription factor 1 (HSF1). In the present study, we examined the effects of medium osmolarity on both the activation of HSF1 and the programmed cell death in normal human fibroblasts during cellular senescence. The activation of HSF1 occurred rapidly in presenescent (early passage) IMR-90 cells when exposed to either hypo-osmotic or hyperosmotic stress. In contrast, the activation of HSF1 was significantly attenuated in senescent cells. Western blot analysis indicated that equal amounts of HSF1 were present as monomers in the cytoplasm of both presenescent and senescent cells in normal growth medium. Under either hypo-osmotic or hyperosmotic stress, trimerization and nuclear localization of HSF1 occurred in presenescent cells but not in senescent cells. More than 80% of HSF1 in senescent cells remained as monomers in the cytoplasm under osmotic stress, suggesting a defect in the signal transduction pathways that lead to HSF1 trimerization or a dysfunction in the HSF1 protein itself. Possible involvement of mitogen-activated protein kinase (MAPK) signal transduction pathways in the activation HSF1 was investigated by monitoring the activation of the three MAPKs, ERK1/2, JNK1/2, and p38, in cells exposed to hypo-osmotic or hyperosmotic stress. All three MAPKs were activated by hyperosmotic stress but not hypo-osmotic stress, suggesting that the MAPK signal transduction pathways may not be directly linked to the osmotic stress-induced activation of HSF1. In contrast to the rapid heat shock transcription factor (HSF) activation, apoptosis occurred only after long-term exposure to hypo-osmotic or hyperosmotic stress. Despite the prominent induction of HSF1 activation, the presenescent cells were more sensitive than the senescent cells to the osmotic stress-induced apoptosis.

In vivo activation of neural heat shock transcription factor HSF1 by a physiologically relevant increase in body temperature

Molecular mechanisms which underlie the heat shock response have commonly been analyzed using tissue culture systems, with less investigation of the intact mammal. In tissue culture, a temperature elevation of 5°C is required to activate mammalian heat shock transcription factor 1 (HSF1) to the DNA-binding form. We demonstrate that a physiologically relevant increase in body temperature of 2.5 ± 0.2°C, similar to that attained during fever reactions, is sufficient to activate HSF1 in the rabbit nervous system. Maximal HSF activation, as measured by gel mobility shift assay, was attained at 1 hr with the cerebellum showing the strongest signal. Supershift experiments with antibodies specific to HSF1 and HSF2 demonstrated that the signal reflected activation of HSF1. Western blot analysis showed that cerebellum exhibited high levels of HSF1 protein. © 1996 Wiley-Liss, Inc.