Targeted Replacement of HSF1 Phosphorylation Sites at S303/S307 with Alanine Residues in Mice Increases Cell Proliferation and Drug Resistance.

Mammalian heat shock factor HSF1 transcriptional activity is controlled by a multitude of phosphorylations that occur under physiological conditions or following exposure of cells to a variety of stresses. One set of HSF1 phosphorylation is on serine 303 and serine 307 (S303/S307). These HSF1 phosphorylation sites are known to repress its transcriptional activity. Here, we describe a knock-in mouse model where these two serine residues were replaced by alanine residues and have determined the impact of these mutations on cellular proliferation and drug resistance. Our previous study using this mouse model indicated the susceptibility of the mutant mice to become obese with age due to an increase in basal levels of heat shock proteins (HSPs) and chronic inflammation. Since HSF1 transcriptional activity is increased in many tumor types, this mouse model may be a useful tool for studies related to cellular transformation and cancer.

Dusp26 phosphatase regulates mitochondrial respiration and oxidative stress and protects neuronal cell death.

The dual specificity protein phosphatases (Dusps) control dephosphorylation of mitogen-activated protein kinases (MAPKs) as well as other substrates. Here, we report that Dusp26, which is highly expressed in neuroblastoma cells and primary neurons is targeted to the mitochondrial outer membrane via its NH2-terminal mitochondrial targeting sequence. Loss of Dusp26 has a significant impact on mitochondrial function that is associated with increased levels of reactive oxygen species (ROS), reduction in ATP generation, reduction in mitochondria motility and release of mitochondrial HtrA2 protease into the cytoplasm. The mitochondrial dysregulation in dusp26-deficient neuroblastoma cells leads to the inhibition of cell proliferation and cell death. In vivo, Dusp26 is highly expressed in neurons in different brain regions, including cortex and midbrain (MB). Ablation of Dusp26 in mouse model leads to dopaminergic (DA) neuronal cell loss in the substantia nigra par compacta (SNpc), inflammatory response in MB and striatum, and phenotypes that are normally associated with Neurodegenerative diseases. Consistent with the data from our mouse model, Dusp26 expressing cells are significantly reduced in the SNpc of Parkinson's Disease patients. The underlying mechanism of DA neuronal death is that loss of Dusp26 in neurons increases mitochondrial ROS and concurrent activation of MAPK/p38 signaling pathway and inflammatory response. Our results suggest that regulation of mitochondrial-associated protein phosphorylation is essential for the maintenance of mitochondrial homeostasis and dysregulation of this process may contribute to the initiation and development of neurodegenerative diseases.

HSF1-Mediated Control of Cellular Energy Metabolism and mTORC1 Activation Drive Acute T-Cell Lymphoblastic Leukemia Progression.

Deregulated oncogenic signaling linked to PI3K/AKT and mTORC1 pathway activation is a hallmark of human T-cell acute leukemia (T-ALL) pathogenesis and contributes to leukemic cell resistance and adverse prognosis. Notably, although the multiagent chemotherapy of leukemia leads to a high rate of complete remission, options for salvage therapy for relapsed/refractory disease are limited due to the serious side effects of augmenting cytotoxic chemotherapy. We report that ablation of HSF1, a key transcriptional regulator of the chaperone response and cellular bioenergetics, from mouse T-ALL tumors driven by PTEN loss or human T-ALL cell lines, has significant therapeutic effects in reducing tumor burden and sensitizing malignant cell death. From a mechanistic perspective, the enhanced sensitivity of T-ALLs to HSF1 depletion resides in the reduced MAPK-ERK signaling and metabolic and ATP-producing capacity of malignant cells lacking HSF1 activity. Impaired mitochondrial ATP production and decreased intracellular amino acid content in HSF1-deficient T-ALL cells trigger an energy-saving adaptive response featured by attenuation of the mTORC1 activity, which is coregulated by ATP, and its downstream target proteins (p70S6K and 4E-BP). This leads to protein translation attenuation that diminishes oncogenic signals and malignant cell growth. Collectively, these metabolic alterations in the absence of HSF1 activity reveal cancer cell liabilities and have a profound negative impact on T-ALL progression. IMPLICATIONS: Targeting HSF1 and HSF1-dependent cancer-specific anabolic and protein homeostasis programs has a significant therapeutic potential for T-ALL and may prevent progression of relapsed/refractory disease.

The Molecular Chaperone Heat Shock Protein 70 Controls Liver Cancer Initiation and Progression by Regulating Adaptive DNA Damage and Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase Signaling Pathways.

Delineating the mechanisms that drive hepatic injury and hepatocellular carcinoma (HCC) progression is critical for development of novel treatments for recurrent and advanced HCC but also for the development of diagnostic and preventive strategies. Heat shock protein 70 (HSP70) acts in concert with several cochaperones and nucleotide exchange factors and plays an essential role in protein quality control that increases survival by protecting cells against environmental stressors. Specifically, the HSP70-mediated response has been implicated in the pathogenesis of cancer, but the specific mechanisms by which HSP70 may support malignant cell transformation remains to be fully elucidated. Here, we show that genetic ablation of HSP70 markedly impairs HCC initiation and progression by distinct but overlapping pathways. This includes the potentiation of the carcinogen-induced DNA damage response, at the tumor initiation stage, to increase the p53-dependent surveillance response leading to the cell cycle exit or death of genomically damaged differentiated pericentral hepatocytes, and this may also prevent their conversion into more proliferating HCC progenitor cells. Subsequently, activation of a mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) negative feedback pathway diminishes oncogenic signals, thereby attenuating premalignant cell transformation and tumor progression. Modulation of HSP70 function may be a strategy for interfering with oncogenic signals driving liver cell transformation and tumor progression, thus providing an opportunity for human cancer control.

The transcriptional regulator of the chaperone response HSF1 controls hepatic bioenergetics and protein homeostasis.

Metabolic energy reprogramming facilitates adaptations to a variety of stress conditions and cellular dysfunction, but how the energetic demands are monitored and met in response to physiological stimuli remains elusive. Our data support a model demonstrating that heat shock factor 1 (HSF1), a master transcriptional regulator of the chaperone response, has been coopted from its role as a critical protein quality-control regulator to having a central role in systemic energy sensing and for metabolic adaptation to nutrient availability. We found that in the absence of HSF1, levels of NAD+ and ATP are not efficiently sustained in hepatic cells, largely because of transcriptional repression of nicotinamide phosphoribosyltransferase in the NAD+ salvage pathway. Mechanistically, the defect in NAD+ and ATP synthesis linked to a loss of NAD+-dependent deacetylase activity, increased protein acetylation, and impaired mitochondrial integrity. Remarkably, the drop in ATP level caused by HSF1 loss invoked an adaptive response featuring the inhibition of energetically demanding processes, including gluconeogenesis, translation, and lipid synthesis. Our work identifies HSF1 as a central regulator of cellular bioenergetics and protein homeostasis that benefits malignant cell progression and exacerbates development of metabolic diseases.

Therapeutic inducers of the HSP70/HSP110 protect mice against traumatic brain injury.

Traumatic brain injury (TBI) induces severe harm and disability in many accident victims and combat-related activities. The heat-shock proteins Hsp70/Hsp110 protect cells against death and ischemic damage. In this study, we used mice deficient in Hsp110 or Hsp70 to examine their potential requirement following TBI. Data indicate that loss of Hsp110 or Hsp70 increases brain injury and death of neurons. One of the mechanisms underlying the increased cell death observed in the absence of Hsp110 and Hsp70 following TBI is the increased expression of reactive oxygen species-induced p53 target genes Pig1, Pig8, and Pig12. To examine whether drugs that increase the levels of Hsp70/Hsp110 can protect cells against TBI, we subjected mice to TBI and administered Celastrol or BGP-15. In contrast to Hsp110- or Hsp70i-deficient mice that were not protected following TBI and Celastrol treatment, there was a significant improvement of wild-type mice following administration of these drugs during the first week following TBI. In addition, assessment of neurological injury shows significant improvement in contextual and cued fear conditioning tests and beam balance in wild-type mice that were treated with Celastrol or BGP-15 following TBI compared to TBI-treated mice. These studies indicate a significant role of Hsp70/Hsp110 in neuronal survival following TBI and the beneficial effects of Hsp70/Hsp110 inducers toward reducing the pathological consequences of TBI. Our data indicate that loss of Hsp110 or Hsp70 in mice increases brain injury following TBI. (a) One of the mechanisms underlying the increased cell death observed in the absence of these Hsps following TBI is the increased expression of ROS-induced p53 target genes known as Pigs. In addition, (b) using drugs (Celastrol or BGP-15) to increase Hsp70/Hsp110 levels protect cells against TBI, suggesting the beneficial effects of Hsp70/Hsp110 inducers to reduce the pathological consequences of TBI.

An essential role for heat shock transcription factor binding protein 1 (HSBP1) during early embryonic development.

Heat shock factor binding protein 1 (HSBP1) is a 76 amino acid polypeptide that contains two arrays of hydrophobic heptad repeats and was originally identified through its interaction with the oligomerization domain of heat shock factor 1 (Hsf1), suppressing Hsf1's transcriptional activity following stress. To examine the function of HSBP1 in vivo, we generated mice with targeted disruption of the hsbp1 gene and examined zebrafish embryos treated with HSBP1-specific morpholino oligonucleotides. Our results show that hsbp1 is critical for preimplantation embryonic development. Embryonic stem (ES) cells deficient in hsbp1 survive and proliferate normally into the neural lineage in vitro; however, lack of hsbp1 in embryoid bodies (EBs) leads to disorganization of the germ layers and a reduction in the endoderm-specific markers (such as α-fetoprotein). We further show that hsbp1-deficient mouse EBs and knockdown of HSBP1 in zebrafish leads to an increase in the expression of the neural crest inducers Snail2, Tfap2α and Foxd3, suggesting a potential role for HSBP1 in the Wnt pathway. The hsbp1-deficient ES cells, EBs and zebrafish embryos with reduced HSBP1 levels exhibit elevated levels of Hsf1 activity and expression of heat shock proteins (Hsps). We conclude that HSBP1 plays an essential role during early mouse and zebrafish embryonic development.

Heat shock factor Hsf1 cooperates with ErbB2 (Her2/Neu) protein to promote mammary tumorigenesis and metastasis.

ErbB2/Neu oncogene is overexpressed in 25% of invasive/metastatic breast cancers. We have found that deletion of heat shock factor Hsf1 in mice overexpressing ErbB2/Neu significantly reduces mammary tumorigenesis and metastasis. Hsf1(+/-)ErbB2/Neu(+) tumors exhibit reduced cellular proliferative and invasive properties associated with reduced activated ERK1/2 and reduced epithelial-mesenchymal transition (EMT). Hsf1(+/+)Neu(+) mammary epithelial cells exposed to TGFß show high levels of ERK1/2 activity and EMT; this is associated with reduced expression of E-cadherin and increased expression of Slug and vimentin, a mesenchymal marker. In contrast, Hsf1(-/-)Neu(+) or Hsf1(+/+)Neu(+) cells do not exhibit activated ERK1/2 and show reduced EMT in the presence of TGFß. The ineffective activation of the RAS/RAF/MEK/ERK1/2 signaling pathway in cells with reduced levels of HSF1 is due to the low levels of HSP90 in complex with RAF1 that are required for RAF1 stability and maturation. These results indicate a powerful inhibitory effect conferred by HSF1 downstream target genes in the inhibition of ErbB2-induced breast cancers in the absence of the Hsf1 gene.

Inactivation of heat shock factor Hsf4 induces cellular senescence and suppresses tumorigenesis in vivo.

Studies suggest that Hsf4 expression correlates with its role in cell growth and differentiation. However, the role of Hsf4 in tumorigenesis in vivo remains unexplored. In this article, we provide evidence that absence of the Hsf4 gene suppresses evolution of spontaneous tumors arising in p53- or Arf-deficient mice. Furthermore, deletion of hsf4 alters the tumor spectrum by significantly inhibiting development of lymphomas that are normally observed in the majority of mice lacking p53 or Arf tumor suppressor genes. Using mouse embryo fibroblasts deficient in the hsf4 gene, we have found that these cells exhibit reduced proliferation that is associated with induction of senescence and senescence-associated ß-galactosidase (SA-ß-gal). Cellular senescence in hsf4-deficient cells is associated with the increased expression of the cyclin-dependent kinase inhibitors, p21 and p27 proteins. Consistent with the cellular senescence observed in vitro, specific normal tissues of hsf4(-/-) mice and tumors that arose in mice deficient in both hsf4 and p53 genes exhibit increased SA-ß-gal activity and elevated levels of p27 compared with wild-type mice. These results suggest that hsf4 deletion-induced senescence is also present in vivo. Our results therefore indicate that Hsf4 is involved in modulation of cellular senescence, which can be exploited during cancer therapy.

Heat shock transcription factor 1 is a key determinant of HCC development by regulating hepatic steatosis and metabolic syndrome.

Hepatocellular carcinoma (HCC) occurrence and progression are linked tightly to progressive hepatic metabolic syndrome associated with insulin resistance, hepatic steatosis, and chronic inflammation. Heat shock transcription factor 1 (HSF1), a major transactivator of stress proteins, increases survival by protecting cells against environmental stressors. It has been implicated in the pathogenesis of cancer, but specific mechanisms by which HSF1 supports cancer development remain elusive. We propose a pathogenic mechanism whereby HSF1 activation promotes growth of premalignant cells and HCC development by stimulating lipid biosynthesis and perpetuating chronic hepatic metabolic disease induced by carcinogens. Our work shows that inactivation of HSF1 impairs cancer progression, mitigating adverse effects of carcinogens on hepatic metabolism by enhancing insulin sensitivity and sensitizing activation of AMP-activated protein kinase (AMPK), an important regulator of energy homeostasis and inhibitor of lipid synthesis. HSF1 is a potential target for the control of hepatic steatosis, hepatic insulin resistance, and HCC development.

Heat shock factor 1 deficiency via its downstream target gene alphaB-crystallin (Hspb5) impairs p53 degradation.

Heat shock factor Hsf1 regulates the stress-inducibility of heat shock proteins (Hsps) or molecular chaperones. One of the functions attributed to Hsps is their participation in folding and degradation of proteins. We recently showed that hsf1(-/-) cells accumulate ubiquitinated proteins. However, a direct role for Hsf1 in stability of specific proteins such as p53 has not been elucidated. We present evidence that cells deficient in hsf1 accumulate wild-type p53 protein. We further show that hsf1(-/-) cells express lower levels of alphaB-crystallin and cells deficient in alphaB-crystallin also accumulate p53 protein. Reports indicate that alphaB-crystallin binds to Fbx4 ubiquitin ligase, and they target cyclin D1 for degradation through a pathway involving the SCF (Skp1-Cul1-F-box) complex. Towards determining a mechanism for p53 degradation involving alphaB-crystallin and Hsf1, we have found that ectopic expression of Fbx4 in wild-type mouse embryo fibroblasts (MEFs) expressing mutant p53 (p53R175H) leads to increase in its degradation, while MEFs deficient in hsf1 or alphaBcry are defective in degradation of this p53 protein. In addition, immunoprecipitated p53R175H from wild-type MEFs is able to pull-down both alphaB-crystallin and Fbx4. Finally, immunoprecipitated wild-type p53 from doxorubicin treated U2OS cells can pull-down endogenous alphaB-crystallin and Fbx4. These results indicate that hsf1- and alphaBcry-deficient cells accumulate p53 due to reduced levels of alphaB-crystallin in these cells. Elevated levels of p53 in hsf1- and alphaBcry-deficient cells lead to their increased sensitivity to DNA damaging agents. These data reveal a novel mechanism for protein degradation through Hsf1 and alphaB-crystallin.

Regulation of immune response and inflammatory reactions against viral infection by VCAM-1.

The migration of activated antigen-specific immune cells to the target tissues of virus replication is controlled by the expression of adhesion molecules on the vascular endothelium that bind to ligands on circulating lymphocytes. Here, we demonstrate that the adhesion pathway mediated by vascular cell adhesion molecule 1 (VCAM-1) plays a role in regulating T-cell-mediated inflammation and pathology in nonlymphoid tissues, including the central nervous system (CNS) during viral infection. The ablation of VCAM-1 expression from endothelial and hematopoietic cells using a loxP-Cre recombination strategy had no major effect on the induction or overall tissue distribution of antigen-specific T cells during a systemic infection with lymphocytic choriomeningitis virus (LCMV), except in the case of lung tissue. However, enhanced resistance to lethal LCM and the significantly reduced magnitude and duration of footpad swelling observed in VCAM-1 mutant mice compared to B6 controls suggest a significant role for VCAM-1 in promoting successful local inflammatory reactions associated with efficient viral clearance and even life-threatening immunopathology under particular infection conditions. Interestingly, analysis of the infiltrating populations in the brains of intracerebrally infected mice revealed that VCAM-1 deletion significantly delayed migration into the CNS of antigen-presenting cells (macrophages and dendritic cells), which are critical for optimal stimulation of migrating virus-specific CD8(+) T cells initiating a pathological cascade. We propose that the impaired migration of these accessory cells in the brain may explain the improved clinical outcome of infection in VCAM-1 mutant mice. Thus, these results underscore the potential role of VCAM-1 in regulating the immune response and inflammatory reactions against viral infections.

Control of virus-specific CD8+ T-cell exhaustion and immune-mediated pathology by E3 ubiquitin ligase Cbl-b during chronic viral infection.

A characteristic feature in the immune response to many persistent viral infections is the dysfunction or deletion of antigen-specific T cells (exhaustion). This down-regulation of virus-specific T-cell response represents a critical control mechanism that exists within T-cell activation pathways to prevent lethal disease by inappropriate responses against disseminating virus infections. However, the molecular mechanisms by which the immune system determines whether to mount a full response to such infections remain largely unexplored. Here, we have established that in the murine lymphocytic choriomeningitis virus (LCMV) model, induction of the T-cell receptor signaling inhibitor molecule E3 ligase Cbl-b is critically involved in this decision. In particular, our data revealed that Cbl-b controls the program responsible for T-cell tolerance (exhaustion) induction during a chronic viral infection. Thus, Cbl-b(-/-) mice infected with a low dose of LCMV Docile mount a strong CD8(+) T-cell response that rapidly clears the infection, and the animals remain healthy; in contrast, down-regulation of the epitope-specific CD8(+) T-cell population in persistently infected Cbl-b(-/-) mice, compared to that in chronically infected B6 mice, was significantly delayed, and this was associated with increased morbidity and eventual death in nearly 20% of the animals. Interestingly, infection of Cbl-b(-/-) mice with a moderate virus dose resulted in rapid death with 100% mortality by 7 to 8 days after infection, caused by a dysregulated antiviral T-cell response, whereas the infected B6 mice survived and remained healthy. In conclusion, our results suggest that Cbl-b is critically involved in T-cell exhaustion and prevention of lethal disease.

Insights into function and regulation of small heat shock protein 25 (HSPB1) in a mouse model with targeted gene disruption.

The mammalian small heat shock protein (sHSPs) family is comprised of 10 members and includes HSPB1, which is proposed to play an essential role in cellular physiology, acting as a molecular chaperone to regulate diverse cellular processes. Whilst differential roles for sHSPs are suggested for specific tissues, the relative contribution of individual sHSP family members in cellular and organ physiology remains unclear. To address the function of HSPB1 in vivo and determine its tissue-specific expression during development and in the adult, we generated knock-in mice where the coding sequence of hspb1 is replaced by a lacZ reporter gene. Hspb1 expression marks myogenic differentiation with specific expression first confined to developing cardiac muscles and the vascular system, and later in skeletal muscles with specific expression at advanced stages of myoblast differentiation. In the adult, hspb1 expression was observed in other tissues, such as stratified squamous epithelium of skin, oronasal cavity, tongue, esophagus, and uterine cervix but its expression was most prominent in the musculature. Interestingly, in cardiac muscle hsbp1 expression was down-regulated during the neonatal period and maintained to a relatively low steady-level throughout adulthood. Despite this widespread expression, hspb1-/- mice were viable and fertile with no apparent morphological abnormalities in tissues under physiological conditions. However, at the cellular level and under stress conditions (heat challenge), HSPB1 act synergistically with the stress-induced HSPA1 (HSP70) in thermotolerance development, protecting cells from apoptosis. Our data thus indicate a nonessential role for HSPB1 in embryonic development and for maintenance of tissues under physiological conditions, but also shows that it plays an important role by acting synergistically with other HSPs during stress conditions to exert cytoprotection and anti-apoptotic effects.

Perforin and Fas cytolytic pathways coordinately shape the selection and diversity of CD8+-T-cell escape variants of influenza virus.

Antigenic variation is a viral strategy exploited to promote survival in the face of the host immune response and represents a major challenge for efficient vaccine development. Influenza viruses are pathogens with high transmissibility and mutation rates, enabling viral escape from immunity induced by prior infection or vaccination. Intense selection from neutralizing antibody drives antigenic changes in the surface glycoproteins, resulting in emergence of new strains able to reinfect hosts immune to previously circulating viruses. CD8+ cytotoxic T cells (CTLs) also provide protective immunity from influenza virus infection and may contribute to the antigenic evolution of influenza viruses. Utilizing mice transgenic for an influenza virus NP366-374 peptide-specific T-cell receptor, we demonstrated that the respiratory tract is a suitable site for generation of escape variants of influenza virus selected by CTL in vivo. In this report the contributions of the perforin and Fas pathways utilized by influenza virus-specific CTLs in viral clearance and selection of CTL escape variants have been evaluated. While transgenic CTLs deficient in either perforin- or Fas-mediated pathways are efficient in initial pulmonary viral control, variant virus emergence was observed in all the mice studied, although the spectrum of viral CTL escape variants selected varied profoundly. Thus, a less-restricted repertoire of escape variants was observed in mice with an intact perforin cytotoxic pathway compared with a limited variant diversity in perforin pathway-deficient mice, although maximal variant diversity was observed in mice having both Fas and perforin pathways intact. We conclude that selection of viral CTL escape variants reflects coordinate action between the tightly controlled perforin/granzyme pathway and the more promiscuous Fas/FasL pathway.

Differential tissue-specific regulation of antiviral CD8+ T-cell immune responses during chronic viral infection.

The hallmarks of the immune response to viral infections are the expansion of antigen-specific CD8(+) cytotoxic T lymphocytes (CTLs) after they encounter antigen-presenting cells in the lymphoid tissues and their subsequent redistribution to nonlymphoid tissues to deal with the pathogen. Control mechanisms exist within CTL activation pathways to prevent inappropriate CTL responses against disseminating infections with a broad distribution of pathogen in host tissues. This is demonstrated during overwhelming infection with the noncytolytic murine lymphocytic choriomeningitis virus, in which clonal exhaustion (anergy and/or deletion) of CTLs prevents immune-mediated pathology but allows persistence of the virus. The mechanism by which the immune system determines whether or not to mount a full response to such infections is unknown. Here we present data showing that the initial encounter of specific CTLs with infected cells in lymphoid tissues is critical for this decision. Whether the course of the viral infection is acute or persistent for life primarily depends on the degree and kinetics of CTL exhaustion in infected lymphoid tissues. Virus-driven CTL expansion in lymphoid tissues resulted in the migration of large quantities of CTLs to nonlymphoid tissues, where they persisted at stable levels. Surprisingly, although virus-specific CTLs were rapidly clonally exhausted in lymphoid tissues under conditions of chronic infection, a substantial number of them migrated to nonlymphoid tissues, where they retained an effector phenotype for a long time. However, these cells were unable to control the infection and progressively lost their antiviral capacities (cytotoxicity and cytokine secretion) in a hierarchical manner before their eventual physical elimination. These results illustrate the differential tissue-specific regulation of antiviral T-cell responses during chronic infections and may help us to understand the dynamic relationship between antigen and T-cell populations in many persistent infections in humans.

Targeted disruption of hsf1 leads to lack of thermotolerance and defines tissue-specific regulation for stress-inducible Hsp molecular chaperones.

The rapid synthesis of heat shock proteins (Hsps) in cells subjected to environmental challenge is controlled by heat shock transcription factor-1 (Hsf1). Regulation of Hsps by Hsf1 is highly complex and, in the whole organism, remains largely unexplored. In this study, we have used mouse embryo fibroblasts and bone marrow progenitor cells from hsf1-/- mice as well as hsp70.3-lacZ knock-in mice bred on the hsf1deficient genetic background (hsf1-/--hsp70.3+/--lacZ), to further elucidate the function of Hsf1 and its participation as a transcriptional activator of Hsp70 synthesis under normal or heat-induced stress conditions in vitro and in vivo. The results revealed that heat-induced Hsp70 expression in mouse tissue is entirely controlled by Hsf1, whereas its activity is not required for tissue-specific constitutive Hsp70 expression. We further demonstrate that Hsf1 is critical for maintaining cellular integrity after heat stress and that cells from hsf1-/- mice lack the ability to develop thermotolerance. This deficiency is explained by the elimination of stress-inducible Hsp70 and Hsp25 response in the absence of Hsf1 activity, leading to a lack of Hsp-mediated inhibition of apoptotic cell death via both caspase-dependent and caspase-independent pathways. The pivotal role of the Hsf1 transactivator in regulating rapid synthesis of Hsps as a critical cellular defense mechanism against environmental stress-induced damage is underlined.

Critical role for perforin-, Fas/FasL-, and TNFR1-mediated cytotoxic pathways in down-regulation of antigen-specific T cells during persistent viral infection.

Viral persistence following infection with invasive strains of lymphocytic choriomeningitis virus (LCMV) can be achieved by selective down-regulation of virus-specific T lymphocytes. High viral burden in the onset of infection drives responding cells into functional unresponsiveness (anergy) that can be followed by their physical elimination. In this report, we studied down-regulation of the virus-specific CD8(+)-T-cell response during persistent infection of adult mice with LCMV, with emphasis on the role of perforin-, Fas/FasL-, or tumor necrosis factor receptor 1 (TNFR1)-mediated cytolysis in regulating T-cell homeostasis. The results reveal that the absence of perforin, Fas-ligand, or TNFR1 has no significant effect on the kinetics of proliferation and functional inactivation of virus-specific CD8(+) T cells in the onset of chronic LCMV infection. However, these molecules play a critical role in the homeostatic regulation of T cells, influencing the longevity of the virus-specific CD8(+)-T-cell population once it has become anergic. Thus, CD8(+) T cells specific to the dominant LCMV NP(396-404) epitope persist in an anergic state for at least 70 days in perforin-, FasL-, or TNFR1-deficient mice, but they were eliminated by day 30 in C57BL/6 controls. These effects were additive as shown by a deficit of apoptotic death of NP(396-404) peptide-specific CD8(+) T cells in mice lacking both perforin and TNFR1. This suggests a role for perforin-, FasL-, and TNFR1-mediated pathways in down-regulation of the antiviral T cell response during persistent viral infection by determining the fate of antigen-specific T cells. Moreover, virus-specific anergic CD8(+) T cells in persistently infected C57BL/6 mice contain higher levels of Bcl-2 and Bcl-XL than functionally intact T cells generated during acute LCMV infection. In the case of proapoptotic factors, Bax expression did not differ between T-cell populations and Bad was below the limit of detection in all samples. As expression of the Bcl-2 family members controls susceptibility to apoptosis, this finding may provide a molecular basis for the survival of anergic cells under conditions of prolonged antigen stimulation.