Bioactive TNIIIA2 Sequence in Tenascin-C Is Responsible for Macrophage Foam Cell Transformation; Potential of FNIII14 Peptide Derived from Fibronectin in Suppression of Atherosclerotic Plaque Formation.

One of the extracellular matrix proteins, tenascin-C (TN-C), is known to be upregulated in age-related inflammatory diseases such as cancer and cardiovascular diseases. Expression of this molecule is frequently detected, especially in the macrophage-rich areas of atherosclerotic lesions; however, the role of TN-C in mechanisms underlying the progression of atherosclerosis remains obscure. Previously, we found a hidden bioactive sequence termed TNIIIA2 in the TN-C molecule and reported that the exposure of this sequence would be carried out through limited digestion of TN-C by inflammatory proteases. Thus, we hypothesized that some pro-atherosclerotic phenotypes might be elicited from macrophages when they were stimulated by TNIIIA2. In this study, TNIIIA2 showed the ability to accelerate intracellular lipid accumulation in macrophages. In this experimental condition, an elevation of phagocytic activity was observed, accompanied by a decrease in the expression of transporters responsible for lipid efflux. All these observations were mediated through the induction of excessive ß1-integrin activation, which is a characteristic property of the TNIIIA2 sequence. Finally, we demonstrated that the injection of a drug that targets TNIIIA2's bioactivity could rescue mice from atherosclerotic plaque expansion. From these observations, it was shown that TN-C works as a pro-atherosclerotic molecule through an internal TNIIIA2 sequence. The possible advantages of clinical strategies targeting TNIIIA2 are also indicated.

Heat shock-induced heme oxygenase-1 expression in a mouse hepatoma cell line is dependent on HSF1 and modified by NRF2 and BACH1.

The induction mechanism of heme oxygenase-1 (HO-1) by heat shock (HS) is still unknown. Here, we discovered that HS activates the HO-1 expression in a mouse hepatoma cell line (Hepa 1-6). Knockdown experiments showed that the HS-induced HO-1 expression was dependent on HS factor 1 (HSF1). A chromatin immunoprecipitation (ChIP) assay demonstrated that the HS-activated HSF1 bound to the HS elements (HSEs) in the upstream enhancer 1 region (E1). Unexpectedly, HS also facilitates the BTB and CNC homology 1 (BACH1) binding to the Maf recognition elements (MAREs) in E1. We examined the effects of a catalytically inactive CRISPR-associated 9 nucleases (dCas9) with short guide RNAs (sgRNAs), and demonstrated that the HSF1 binding to HSEs in E1 was indispensable for the HS-induced HO-1 expression. Heme treatment (HA) dissociates BACH1 from MAREs and facilitated the binding of nuclear factor-erythroid-2-related factor 2 (NRF2) to MAREs. Following treatment with both HS and HA, the HO-1 induction and the HSF1 binding to HSEs in E1 were most notably observed. These results indicate that the HS-induced HO-1 expression is dependent on the HSF1 binding to HSEs in E1, although modulated by the BACH1 and NRF2 binding to MAREs within the same E1.

Heme oxygenase-1 induction by heat shock in rat hepatoma cell line is regulated by the coordinated function of HSF1, NRF2 and BACH1.

The mechanism of heme oxygenase-1 (HO-1) induction by heat shock (HS) loading remains unclear. Here, we investigated the contribution of transcription factors to HS-induced HO-1 expression, using a rat hepatoma cell line (H-4-II-E). Our results demonstrated that HS treatment resulted in a marked induction of HO-1. Immunohistochemical analysis showed a slight mismatch in the expression levels of HO-1 and HSP70 by HS among cells, suggesting a conflict between multiple induction mechanisms. We observed HS-induced nuclear localization of, not only phosphorylated HSF1 but also NRF2, which is a typical transcription factor activated by oxidative stress. HSF1 knockdown in H-4-II-E markedly reduced HO-1 induction by HS, while NRF2 knockdown resulted in a partial effect. The chromatin immunoprecipitation assay demonstrated that HS loading resulted in significant binding of HSF1 to the HSE in the promoter proximal region of HO-1 gene and another HSE located close to the Maf recognition element (MARE) in the -4 kb upstream enhancer region 1, where NRF2 also bound, together with basic leucine zipper transcription factor 1, a negative transcription factor of HO-1. These observations indicate that HO-1 induction by HS is mainly mediated by HSF1 binding to the proximal HSE. NRF2 binding to MARE by HS is predominantly suppressed by an increased binding of BACH1.

Local redox environment beneath biological membranes probed by palmitoylated-roGFP.

Production of reactive oxygen species (ROS) and consequent glutathione oxidation are associated with various physiological processes and diseases, including cell differentiation, senescence, and inflammation. GFP-based redox sensors provide a straight-forward approach to monitor ROS levels and glutathione oxidation within a living cell at the subcellular resolution. We utilized palmitoylated versions of cytosolic glutathione and hydrogen peroxide sensors (Grx1-roGFP2 and roGFP2-Orp1, respectively) and demonstrated a unique redox environment near biological membranes. In HeLa cells, cytosolic glutathione was practically completely reduced (EGSH/GSSG = - 333mV) and hydrogen peroxide level was under the detectable range. In contrast, the cytoplasmic milieu near membranes of intracellular vesicles exhibited significant glutathione oxidation (EGSH/GSSG > - 256mV) and relatively high H2O2 production, which was not observed for the plasma membrane. These vesicles colocalized with internalized EGFR, suggesting that H2O2 production and glutathione oxidation are characteristics of cytoplasmic surfaces of the endocytosed vesicles. The results visually illustrate local redox heterogeneity within the cytosol for the first time.

Prevention of Barrier Disruption by Heme Oxygenase-1 in Intestinal Bleeding Model.

In this study we investigated the effect of free heme, the local level of which was increased by bleeding, on the intestinal barrier function, using human epithelial colorectal adenocarcinoma cells (Caco-2). Our results show that the addition of hemin to the culture medium markedly disrupted the barrier function, which was significantly improved by glutamine supplementation. Although hemin treatment caused the increased expression of heme oxygenase (HO)-1, the inhibition of HO activity resulted in the aggravation of hemin-induced barrier dysfunction. Up-regulation of HO-1 by pretreatment with a low concentration of hemin almost completely prevented hemin-induced barrier dysfunction. Taken together, these observations indicate that an abnormally high level of intracellular free heme causes barrier dysfunction, probably through the modulation of proteins forming tight junctions.

Chicken IL-6 is a heat-shock gene.

The febrile response is elicited by pyrogenic cytokines including IL-6 in response to microorganism infections and diseases in vertebrates. Mammalian HSF1, which senses elevations in temperature, negatively regulates the response by suppressing pyrogenic cytokine expression. We here showed that HSF3, an avian ortholog of mammalian HSF1, directly binds to and activates IL-6 during heat shock in chicken cells. Other components of the febrile response mechanism, such as IL-1ß and ATF3, were also differently regulated in mammalian and chicken cells. These results suggest that the febrile response is exacerbated by a feed-forward circuit composed of the HSF3-IL-6 pathway in birds.

Glutamine protects intestinal barrier function of colon epithelial cells from ethanol by modulating Hsp70 expression.

In this study, we investigated the protective effect of glutamine on barrier dysfunction induced by ethanol, by using human epithelial colorectal adenocarcinoma cells (Caco-2). Our results show that addition of glutamine to culture medium significantly improved the disruption of integrity caused by ethanol, which was associated with increased expression of heat shock protein 70 (Hsp70). Ethanol exposure moderately activates heat shock factor 1 (HSF1), which was characterized by increased DNA-binding activity and phosphorylation status of HSF1. Remarkably, glutamine treatment enhanced ethanol-mediated expression of Hsp70 and activation of HSF1. Up-regulation of Hsp70 by pretreatment with heat stress also promoted recovery from the ethanol-induced barrier dysfunction. Taken together, these observations indicate that glutamine protects the intestinal barrier function in Caco-2 cells, in part by modulating HSF1-mediated Hsp70 expression.

Heat shock factor 2 is required for maintaining proteostasis against febrile-range thermal stress and polyglutamine aggregation.

Heat shock response is characterized by the induction of heat shock proteins (HSPs), which facilitate protein folding, and non-HSP proteins with diverse functions, including protein degradation, and is regulated by heat shock factors (HSFs). HSF1 is a master regulator of HSP expression during heat shock in mammals, as is HSF3 in avians. HSF2 plays roles in development of the brain and reproductive organs. However, the fundamental roles of HSF2 in vertebrate cells have not been identified. Here we find that vertebrate HSF2 is activated during heat shock in the physiological range. HSF2 deficiency reduces threshold for chicken HSF3 or mouse HSF1 activation, resulting in increased HSP expression during mild heat shock. HSF2-null cells are more sensitive to sustained mild heat shock than wild-type cells, associated with the accumulation of ubiquitylated misfolded proteins. Furthermore, loss of HSF2 function increases the accumulation of aggregated polyglutamine protein and shortens the lifespan of R6/2 Huntington's disease mice, partly through αB-crystallin expression. These results identify HSF2 as a major regulator of proteostasis capacity against febrile-range thermal stress and suggest that HSF2 could be a promising therapeutic target for protein-misfolding diseases.

Heat shock transcription factor 1 inhibits expression of IL-6 through activating transcription factor 3.

The febrile response is a complex physiological reaction to disease, including a cytokine-mediated increase in body temperature and the activation of inflammatory systems. Fever has beneficial roles in terms of disease prognosis, partly by suppressing the expression of inflammatory cytokines. However, the molecular mechanisms underlining the fever-mediated suppression of inflammatory gene expression have not been clarified. In this study, we showed that heat shock suppresses LPS-induced expression of IL-6, a major pyrogenic cytokine, in mouse embryonic fibroblasts and macrophages. Heat shock transcription factor 1 (HSF1) activated by heat shock induced the expression of activating transcription factor (ATF) 3, a negative regulator of IL-6, and ATF3 was necessary for heat-mediated suppression of IL-6, indicating a fever-mediated feedback loop consisting of HSF1 and ATF3. A comprehensive analysis of inflammatory gene expression revealed that heat pretreatment suppresses LPS-induced expression of most genes (86%), in part (67%) via ATF3. When HSF1-null and ATF3-null mice were injected with LPS, they expressed much higher levels of IL-6 than wild-type mice, resulting in an exaggerated febrile response. These results demonstrate a novel inhibitory pathway for inflammatory cytokines.

Heat shock transcription factor 1 opens chromatin structure of interleukin-6 promoter to facilitate binding of an activator or a repressor.

Heat shock transcription factor 1 (HSF1) not only regulates expression of heat shock genes in response to elevated temperature, but is also involved in developmental processes by regulating genes such as cytokine genes. However, we did not know how HSF1 regulates non-heat shock genes. Here, we show that constitutive HSF1 binding to the interleukin (IL)-6 promoter is necessary for its maximal induction by lipopolysaccharide (LPS) stimulation in mouse embryo fibroblasts and peritoneal macrophages. Lack of HSF1 inhibited LPS-induced in vivo binding of an activator NF-kappaB and a repressor ATF3 to IL-6 promoter. Neither NF-kappaB nor ATF3 binds to the IL-6 promoter in unstimulated HSF1-null cells even if they were overexpressed. Treatment with histone deacetylase inhibitor or a DNA methylation inhibitor restored LPS-induced IL-6 expression in HSF1-null cells, and histone modification enzymes were recruited on the IL-6 promoter in the presence of HSF1. Consistently, chromatin structure of the IL-6 promoter in the presence of HSF1 was more open than that in its absence. These results indicate that HSF1 partially opens the chromatin structure of the IL-6 promoter for an activator or a repressor to bind to it, and provides a novel mechanism of gene regulation by HSF1.

Maintenance of olfactory neurogenesis requires HSF1, a major heat shock transcription factor in mice.

Heat shock transcription factors (HSFs) play roles not only in heat shock response but also in development of the reproductive organs, brain, and lens. Here, we analyzed sensory organs and found abnormalities of the olfactory epithelium in adult HSF1-null mice, which is developmentally related to the lens. The olfactory epithelium was normal until postnatal 3 weeks but was not maintained later than 4 weeks in HSF1-null mice. The olfactory epithelium was atrophied with increased cell death of olfactory sensory neurons. Analysis of the epithelium revealed that induction of HSP expression and reduction of LIF expression are lacking in adult HSF1-null mice. We found that DNA binding activity of HSF1 is induced in the olfactory epithelium later than 4 weeks and that HSF1 binds directly to Lif gene and inhibits its expression. HSF4 has opposing effects on LIF expression and olfactory neurogenesis. These data indicate that HSF1 is required for the precise expression of Hsp and cytokine genes that is obligatory for maintenance of olfactory neurogenesis in adult mice and suggest that stress-related processes are involved in its maintenance.

Active HSF1 significantly suppresses polyglutamine aggregate formation in cellular and mouse models.

Polyglutamine diseases are inherited neurodegenerative diseases characterized by misfolding and aggregation of proteins possessing expanded polyglutamine repeats. As overexpression of some heat shock protein (Hsp) suppresses polyglutamine aggregates and cell death, it is assumed that combined overexpression of Hsps will suppress that more effectively. Here, we examined the impact of active forms of heat shock transcription factor 1 (HSF1), which induces a set of Hsps, on polyglutamine inclusion formation and disease progression. We found that active HSF1 suppressed polyglutamine inclusion formation more significantly than any combination of Hsps in culture cells, possibly by regulating expression of unknown genes, as well as major Hsps. We crossed R6/2 Huntington disease mice with transgenic mice expressing an active HSF1 (HSF1Tg). Analysis of the skeletal muscle revealed that the polyglutamine inclusion formation and its weight loss were improved in R6/2/HSF1Tg mice. Unexpectedly, the life span of R6/2/HSF1Tg mice was significantly improved, although active HSF1 is not expressed in the brain. These results indicated that active HSF1 has a strong inhibitory effect on polyglutamine aggregate formation in vivo and in vitro.

HSF4 is required for normal cell growth and differentiation during mouse lens development.

The heat shock transcription factor (HSF) family consists of three members in mammals and regulates expression of heat shock genes via a heat shock element. HSF1 and HSF2 are required for some developmental processes, but it is unclear how they regulate these processes. To elucidate the mechanisms of developmental regulation by HSFs, we generated mice in which the HSF4 gene is mutated. HSF4-null mice had cataract with abnormal lens fiber cells containing inclusion-like structures, probably due to decreased expression of gamma-crystallin, which maintains protein stability. Furthermore, we found increased proliferation and premature differentiation of the mutant lens epithelial cells, which is associated with increased expression of growth factors, FGF-1, FGF-4, and FGF-7. Unexpectedly, HSF1 competed with HSF4 for the expression of FGFs not only in the lens but also in other tissues. These findings reveal the lens-specific role of HSF4, which activates gamma-crystallin genes, and also indicate that HSF1 and HSF4 are involved in regulating expression of growth factor genes, which are essential for cell growth and differentiation.

Impaired IgG production in mice deficient for heat shock transcription factor 1.

Heat shock factor 1 (HSF1) is a major transactivator of heat shock proteins in response to heat shock, and it is also involved in oogenesis, spermatogenesis, and placental development. However, we do not know the molecular mechanisms controlling developmental processes. In this study, we found that HSF1-null mice exhibited a significant decrease in the T cell-dependent B cell response. When mice were immunized intraperitoneally with sheep red blood cells, the sheep red blood cell-specific IgG production, especially IgG2a production, in HSF1-null mice was about 50% lower than that in wild-type mice at 6 days after the immunization, whereas IgM production was normal. The number of bromodeoxyuridine-incorporated spleen cells in immunized HSF1-null mice was one-third that in immunized wild-type mice, indicating reduced proliferation of the spleen cells. We analyzed levels of cytokines and chemokines in spleen cells and in peritoneal macrophages stimulated with lipopolysaccharide and interferon-gamma and found that expression levels of interleukin-6 and CCL5 were significantly lower in HSF1-null cells than those in wild-type cells. Furthermore, we demonstrated that the IL-6 gene is a direct target gene of HSF1. These results revealed a novel molecular link between HSF1 and a gene related to immune response and inflammation.

Feeding induces expression of heat shock proteins that reduce oxidative stress.

Heat shock proteins (Hsps) are induced in response to various kinds of environmental and physiological stresses. However, it is unclear whether Hsps play roles in protecting cells in the digestive organs against xenobiotic chemicals. Here, we found that feeding induces expression of a set of Hsps specifically in the mouse liver and intestine by activating heat shock transcription factor 1 (HSF1). In the liver, HSF1 is required to suppress toxic effects of electrophiles, which are xenobiotic chemicals causing oxidative stress. We found that overexpression of Hsp27, which elevates cellular glutathione level, promotes survival of culture cells exposed to electrophiles. These results suggest a novel mechanism of cell protection against xenobiotic chemicals in the food.

Hsp25, a member of the Hsp30 family, promotes inclusion formation in response to stress.

Protein aggregates are oligomeric complexes of misfolded proteins, and serve as the seeds of inclusion bodies termed aggresomes in the cells. Heat shock proteins (Hsps) prevent misfolding and aggregate formation. Here, we found that only avian Hsp25 dominantly accumulated in the aggresomes induced by proteasome inhibition. Molecular cloning of chicken Hsp25 (cHsp25) revealed that it belongs to the Hsp30 family, which is a subfamily of the alpha-crystallin/small Hsp gene family. Unexpectedly, overexpression of cHsp25 into HeLa cells promoted inclusion formation whereas overexpression of mouse Hsp27 and its chicken homologue did not. These results suggest that cHsp25 acts differently from other small Hsps on protein aggregates.

Activation of heat shock genes is not necessary for protection by heat shock transcription factor 1 against cell death due to a single exposure to high temperatures.

Heat shock response, which is characterized by the induction of a set of heat shock proteins, is essential for induced thermotolerance and is regulated by heat shock transcription factors (HSFs). Curiously, HSF1 is essential for heat shock response in mammals, whereas in avian HSF3, an avian-specific factor is required for the burst activation of heat shock genes. Amino acid sequences of chicken HSF1 are highly conserved with human HSF1, but those of HSF3 diverge significantly. Here, we demonstrated that chicken HSF1 lost the ability to activate heat shock genes through the amino-terminal domain containing an alanine-rich sequence and a DNA-binding domain. Surprisingly, chicken and human HSF1 but not HSF3 possess a novel function that protects against a single exposure to mild heat shock, which is not mediated through the activation of heat shock genes. Overexpression of HSF1 mutants that could not bind to DNA did not restore the susceptibility to cell death in HSF1-null cells, suggesting that the new protective role of HSF1 is mediated through regulation of unknown target genes other than heat shock genes. These results uncover a novel role of vertebrate HSF1, which has been masked under the roles of heat shock proteins.