Regulation of HSF1 transcriptional complexes under proteotoxic stress: Mechanisms of heat shock gene transcription involve the stress-induced HSF1 complex formation, changes in chromatin states, and formation of phase-separated condensates: Mechanisms of heat shock gene transcription involve the stress-induced HSF1 complex formation, changes in chromatin states, and formation of phase-separated condensates.

Environmental, physiological, and pathological stimuli induce the misfolding of proteins, which results in the formation of aggregates and amyloid fibrils. To cope with proteotoxic stress, cells are equipped with adaptive mechanisms that are accompanied by changes in gene expression. The evolutionarily conserved mechanism called the heat shock response is characterized by the induction of a set of heat shock proteins (HSPs), and is mainly regulated by heat shock transcription factor 1 (HSF1) in mammals. We herein introduce the mechanisms by which HSF1 tightly controls the transcription of HSP genes via the regulation of pre-initiation complex recruitment in their promoters under proteotoxic stress. These mechanisms involve the stress-induced regulation of HSF1-transcription complex formation with a number of coactivators, changes in chromatin states, and the formation of phase-separated condensates through post-translational modifications.

The pericentromeric protein shugoshin 2 cooperates with HSF1 in heat shock response and RNA Pol II recruitment.

The recruitment of RNA polymerase II (Pol II) to core promoters is highly regulated during rapid induction of genes. In response to heat shock, heat shock transcription factor 1 (HSF1) is activated and occupies heat shock gene promoters. Promoter-bound HSF1 recruits general transcription factors and Mediator, which interact with Pol II, but stress-specific mechanisms of Pol II recruitment are unclear. Here, we show in comparative analyses of HSF1 paralogs and their mutants that HSF1 interacts with the pericentromeric adaptor protein shugoshin 2 (SGO2) during heat shock in mouse cells, in a manner dependent on inducible phosphorylation of HSF1 at serine 326, and recruits SGO2 to the HSP70 promoter. SGO2-mediated binding and recruitment of Pol II with a hypophosphorylated C-terminal domain promote expression of HSP70, implicating SGO2 as one of the coactivators that facilitate Pol II recruitment by HSF1. Furthermore, the HSF1-SGO2 complex supports cell survival and maintenance of proteostasis in heat shock conditions. These results exemplify a proteotoxic stress-specific mechanism of Pol II recruitment, which is triggered by phosphorylation of HSF1 during the heat shock response.

Testicular localization of activating transcription factor 1 and its potential function during spermatogenesis†.

Activating transcription factor 1 (ATF1), belonging to the CREB/ATF family of transcription factors, is highly expressed in the testes. However, its role in spermatogenesis has not yet been established. Here, we aimed to elucidate the impact of ATF1 in spermatogenesis by examining the expression pattern of ATF1 in mice and the effect of ATF1 knockdown in the mouse testes. We found that ATF1 is expressed in various organs, with very high levels in the testes. Immunohistochemical staining showed that ATF1 was localized in the nuclei of spermatogonia and co-localized with proliferating cell nuclear antigen. In ATF1-deficient mice, the seminiferous tubules of the testis contained cells at all developmental stages; however, the number of spermatocytes was decreased. Proliferating cell nuclear antigen expression was decreased and apoptotic cells were rare in the seminiferous tubules. These results indicate that ATF1 plays a role in male germ cell proliferation and sperm production.

RPA assists HSF1 access to nucleosomal DNA by recruiting histone chaperone FACT.

Transcription factor access to regulatory elements is prevented by the nucleosome. Heat shock factor 1 (HSF1) is a winged helix transcription factor that plays roles in control and stressed conditions by gaining access to target elements, but mechanisms of HSF1 access are not well known in mammalian cells. Here, we show the physical interaction between the wing motif of human HSF1 and replication protein A (RPA), which is involved in DNA metabolism. Depletion of RPA1 abolishes HSF1 access to the promoter of HSP70 in unstressed condition and delays its rapid activation in response to heat shock. The HSF1-RPA complex leads to preloading of RNA polymerase II and opens the chromatin structure by recruiting a histone chaperone, FACT. Furthermore, this interaction is required for melanoma cell proliferation. These results provide a mechanism of constitutive HSF1 access to nucleosomal DNA, which is important for both basal and inducible gene expression.

A novel, potent dual inhibitor of Arg-gingipains and Lys-gingipain as a promising agent for periodontal disease therapy.

The periodontal pathogen Porphyromonas gingivalis produces a unique class of cysteine proteinases termed gingipains that comprises Arg-gingipain (Rgp) and Lys-gingipain (Kgp). Growing evidence indicates that these 2 types of gingipains synergistically contribute to the entire virulence of the organism and increase the risk of periodontal disease (PD) by disrupting the host immune system and degrading the host tissue and plasma proteins. Therefore, a dual inhibitor of both gingipains would have attractive clinical potential for PD therapy. In this study, a novel, potent, dual inhibitor of Rgp and Kgp was developed through structure-based drug design, and its biological potency was evaluated in vitro and in vivo. This inhibitor had low nanomolar inhibitory potency (Ki=40 nM for Rgp, Ki=0.27 nM for Kgp) and good selectivity for host proteases and exhibited potent antibacterial activity against P. gingivalis by abrogating its manifold pathophysiological functions. The therapeutic potential of this inhibitor in vivo was also verified by suppressing the vascular permeability that was enhanced in guinea pigs by the organism and the gingival inflammation in beagle dog PD models. These findings suggest that a dual inhibitor of Rgp and Kgp would exhibit noteworthy anti-inflammatory activity in the treatment of PD.

Heat shock factor 1 ameliorates proteotoxicity in cooperation with the transcription factor NFAT.

Heat shock transcription factor 1 (HSF1) is an important regulator of protein homeostasis (proteostasis) by controlling the expression of major heat shock proteins (Hsps) that facilitate protein folding. However, it is unclear whether other proteostasis pathways are mediated by HSF1. Here, we identified novel targets of HSF1 in mammalian cells, which suppress the aggregation of polyglutamine (polyQ) protein. Among them, we show that one of the nuclear factor of activated T cells (NFAT) proteins, NFATc2, significantly inhibits polyQ aggregation in cells and is required for HSF1-mediated suppression of polyQ aggregation. NFAT deficiency accelerated disease progression including aggregation of a mutant polyQ-huntingtin protein and shortening of lifespan in R6/2 Huntington's disease mice. Furthermore, we found that HSF1 and NFAT cooperatively induce the expression of the scaffold protein PDZK3 and αB-crystallin, which facilitate the degradation of polyQ protein. These results show the first mechanistic basis for the observation that HSF1 has a much more profound effect on proteostasis than individual Hsp or combination of different Hsps, and suggest a new pathway for ameliorating protein-misfolding diseases.

The Mediator subunit MED12 promotes formation of HSF1 condensates on heat shock response element arrays in heat-shocked cells.

Upon heat shock, activated heat shock transcription factor 1 (HSF1) binds to the heat shock response elements (HSEs) in the promoters of mammalian heat shock protein (HSP)-encoding genes and recruits the preinitiation complex and coactivators, including Mediator. These transcriptional regulators may be concentrated in phase-separated condensates around the promoters, but they are too minute to be characterized in detail. We herein established HSF1-/- mouse embryonic fibroblasts harbouring HSP72-derived multiple HSE arrays and visualized the condensates of fluorescent protein-tagged HSF1 with liquid-like properties upon heat shock. Using this experimental system, we demonstrate that endogenous MED12, a subunit of Mediator, is concentrated in artificial HSF1 condensates upon heat shock. Furthermore, the knockdown of MED12 markedly reduces the size of condensates, suggesting an important role for MED12 in HSF1 condensate formation.

DOCK180 is a Rac activator that regulates cardiovascular development by acting downstream of CXCR4.

RATIONALE: During embryogenesis, the CXC chemokine ligand (CXCL)12 acts on endothelial cells to control cardiac development and angiogenesis. Although biological functions of CXCL12 are exerted in part through activation of the small GTPase Rac, the pathway leading from its receptor CXC chemokine receptor (CXCR)4 to Rac activation remains to be determined. OBJECTIVE: DOCK180 (dedicator of cytokinesis), an atypical Rac activator, has been implicated in various cellular functions. Here, we examined the role of DOCK180 in cardiovascular development. METHODS AND RESULTS: DOCK180 associates with ELMO (engulfment and cell motility) through the N-terminal region containing a Src homology 3 domain. We found that targeted deletion of the Src homology 3 domain of DOCK180 in mice leads to embryonic lethality with marked reduction of DOCK180 expression at the protein level. These mutant mice, as well as DOCK180-deficient mice, exhibited multiple cardiovascular abnormalities resembling those seen in CXCR4-deficient mice. In DOCK180 knocked down endothelial cells, CXCL12-induced Rac activation was impaired, resulting in a marked reduction of cell motility. CONCLUSIONS: These results suggest that DOCK180 links CXCR4 signaling to Rac activation to control endothelial cell migration during cardiovascular development.

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.

HSF1 phosphorylation establishes an active chromatin state via the TRRAP-TIP60 complex and promotes tumorigenesis.

Transcriptional regulation by RNA polymerase II is associated with changes in chromatin structure. Activated and promoter-bound heat shock transcription factor 1 (HSF1) recruits transcriptional co-activators, including histone-modifying enzymes; however, the mechanisms underlying chromatin opening remain unclear. Here, we demonstrate that HSF1 recruits the TRRAP-TIP60 acetyltransferase complex in HSP72 promoter during heat shock in a manner dependent on phosphorylation of HSF1-S419. TRIM33, a bromodomain-containing ubiquitin ligase, is then recruited to the promoter by interactions with HSF1 and a TIP60-mediated acetylation mark, and cooperates with the related factor TRIM24 for mono-ubiquitination of histone H2B on K120. These changes in histone modifications are triggered by phosphorylation of HSF1-S419 via PLK1, and stabilize the HSF1-transcription complex in HSP72 promoter. Furthermore, HSF1-S419 phosphorylation is constitutively enhanced in and promotes proliferation of melanoma cells. Our results provide mechanisms for HSF1 phosphorylation-dependent establishment of an active chromatin status, which is important for tumorigenesis.

DOCK2 is a Rac activator that regulates motility and polarity during neutrophil chemotaxis.

Neutrophils are highly motile leukocytes, and they play important roles in the innate immune response to invading pathogens. Neutrophil chemotaxis requires Rac activation, yet the Rac activators functioning downstream of chemoattractant receptors remain to be determined. We show that DOCK2, which is a mammalian homologue of Caenorhabditis elegans CED-5 and Drosophila melanogaster Myoblast City, regulates motility and polarity during neutrophil chemotaxis. Although DOCK2-deficient neutrophils moved toward the chemoattractant source, they exhibited abnormal migratory behavior with a marked reduction in translocation speed. In DOCK2-deficient neutrophils, chemoattractant-induced activation of both Rac1 and Rac2 were severely impaired, resulting in the loss of polarized accumulation of F-actin and phosphatidylinositol 3,4,5-triphosphate (PIP3) at the leading edge. On the other hand, we found that DOCK2 associates with PIP3 and translocates to the leading edge of chemotaxing neutrophils in a phosphatidylinositol 3-kinase (PI3K)-dependent manner. These results indicate that during neutrophil chemotaxis DOCK2 regulates leading edge formation through PIP3-dependent membrane translocation and Rac activation.

MED12 interacts with the heat-shock transcription factor HSF1 and recruits CDK8 to promote the heat-shock response in mammalian cells.

Activated and promoter-bound heat-shock transcription factor 1 (HSF1) induces RNA polymerase II recruitment upon heat shock, and this is facilitated by the core Mediator in Drosophila and yeast. Another Mediator module, CDK8 kinase module (CKM), consisting of four subunits including MED12 and CDK8, plays a negative or positive role in the regulation of transcription; however, its involvement in HSF1-mediated transcription remains unclear. We herein demonstrated that HSF1 interacted with MED12 and recruited MED12 and CDK8 to the HSP70 promoter during heat shock in mammalian cells. The kinase activity of CDK8 (and its paralog CDK19) promoted HSP70 expression partly by phosphorylating HSF1-S326 and maintained proteostasis capacity. These results indicate an important role for CKM in the protection of cells against proteotoxic stress.

HSF1 is required for induction of mitochondrial chaperones during the mitochondrial unfolded protein response.

The mitochondrial unfolded protein response (UPRmt ) is characterized by the transcriptional induction of mitochondrial chaperone and protease genes in response to impaired mitochondrial proteostasis and is regulated by ATF5 and CHOP in mammalian cells. However, the detailed mechanisms underlying the UPRmt are currently unclear. Here, we show that HSF1 is required for activation of mitochondrial chaperone genes, including HSP60, HSP10, and mtHSP70, in mouse embryonic fibroblasts during inhibition of matrix chaperone TRAP1, protease Lon, or electron transfer complex 1 activity. HSF1 bound constitutively to mitochondrial chaperone gene promoters, and we observed that its occupancy was remarkably enhanced at different levels during the UPRmt . Furthermore, HSF1 supported the maintenance of mitochondrial function under the same conditions. These results demonstrate that HSF1 is required for induction of mitochondrial chaperones during the UPRmt , and thus, it may be one of the guardians of mitochondrial function under conditions of impaired mitochondrial proteostasis.

Inhibition of gingipains prevents Porphyromonas gingivalis-induced preterm birth and fetal death in pregnant mice.

Accumulating epidemiological evidence indicates that infection with Porphyromonas gingivalis which is a major periodontal pathogen, causes preterm birth and low birth weight. However, virulence factors of P. gingivalis responsible for preterm birth/low birth weight remain to be elucidated. In this study, using P. gingivalis-infected pregnant mice as an in vivo model, we investigated whether gingipains-cysteine proteinases produced by P. gingivalis-affect preterm birth and low birth weight. We found that intravenous infection of pregnant mice with P. gingivalis induced higher accumulation of the bacterium in the placenta than that in other organs. Compared to infection with P. gingivalis wild-type, infection with a gingipain-deficient P. gingivalis mutant KDP136 led to significant reduction in preterm birth and pregnancy loss. Although repetitive low-level infections of P. gingivalis failed to induce preterm birth and fetal death, it induced suppressive effects on IFN-γ production. Therapeutically, treatment with ginginpain inhibitors prevented fetal death and preterm birth caused by P. gingivalis infection and resulted in recovery of IFN-γ suppression caused by repetitive chronic P. gingivalis infection. These results indicate that gingipains are major virulence factors of P. gingivalis responsible for preterm birth/low birth, and gingipain inhibitors may be useful not only as a therapeutic agent for periodontal diseases, but also as a preventive medicine for preterm birth/low birth weight.

Poly(ADP-Ribose) Polymerase 1 Promotes the Human Heat Shock Response by Facilitating Heat Shock Transcription Factor 1 Binding to DNA.

The heat shock response (HSR) is characterized by the rapid and robust induction of heat shock proteins (HSPs), including HSP70, in response to heat shock and is regulated by heat shock transcription factor 1 (HSF1) in mammalian cells. Poly(ADP-ribose) polymerase 1 (PARP1), which can form a complex with HSF1 through the scaffold protein PARP13, has been suggested to be involved in the HSR. However, its effects on and the regulatory mechanisms of the HSR are not well understood. Here we show that prior to heat shock, the HSF1-PARP13-PARP1 complex binds to the HSP70 promoter. In response to heat shock, activated and auto-PARylated PARP1 dissociates from HSF1-PARP13 and is redistributed throughout the HSP70 locus. Remarkably, chromatin in the HSP70 promoter is initially PARylated at high levels and decondensed, whereas chromatin in the gene body is moderately PARylated afterwards. Activated HSF1 then binds to the promoter efficiently and promotes the HSR. Chromatin PARylation and HSF1 binding to the promoter are also facilitated by the phosphorylation-dependent dissociation of PARP13. Furthermore, the HSR and proteostasis capacity are reduced by pretreatment with genotoxic stresses, which disrupt the ternary complex. These results illuminate one of the priming mechanisms of the HSR that facilitates the binding of HSF1 to DNA during heat shock.

A role for gingipains in cellular responses and bacterial survival in Porphyromonas gingivalis-infected cells.

Porphyromonas gingivalis is one of the primary etiologic agents of adult periodontitis and is known to produce a unique class of cysteine proteinases, termed gingipains. They consist of Arg-gingipain (Rgp) and Lys-gingipain (Kgp) and exist in the cell-associated and secreted forms. In the current review, we summarize recent knowledge on the pathophysiological role of gingipains in the virulence of P. gingivalis including host cell responses to bacterial infection and its evasion from host defense mechanisms. Studies with various P. gingivalis mutants deficient in Rgp- and/or Kgp-encoding genes and proteinase inhibitors specific for each enzyme have demonstrated that both enzymes play a substantial role in disruption of host defense mechanisms by the bacterium and its survival in vivo. Gingipains are also important in the bacterium-mediated host cell responses and the subsequent intracellular signaling in the infected cells. P. gingivalis can evade the autophagic pathway and instead directly traffic to the endocytic pathway to lysosomes in the infected cells. In addition, gingipains play an important role in acquiring resistance against destruction of the bacterium in the lysosomal system. Furthermore, a major form of the cell-associated gingipain complex composed of the catalytic domains of both enzymes, their adhesin domains, phospholipids, and lipopolysaccharide has recently been isolated and shown to contribute the bacterial evasion of host defense mechanisms and the host tissue breakdown.

Role of Heat Shock Factor 1 in Conserving Cholesterol Transportation in Leydig Cell Steroidogenesis via Steroidogenic Acute Regulatory Protein.

Testicular testosterone synthesis begins with cholesterol transport into mitochondria via steroidogenic acute regulatory (StAR) protein in Leydig cells. Acute heat stress is known to obstruct testicular steroidogenesis by transcriptional repression of StAR. In contrast, chronic heat stress such as cryptorchidism or varicocele generally does not affect testicular steroidogenesis, suggesting that Leydig cells adapt to heat stress and retain their steroid synthesis ability. However, the mechanisms of the stress response in steroid-producing cells are unclear. We examined the relationship between the heat stress response and heat shock factor 1 (HSF1), which protects cells from proteotoxic stress by inducing heat shock protein as a molecular chaperone. The influences of HSF1 deficiency on cholesterol transport by StAR and the expression of steroidogenic enzymes under chronic heat stress were studied in testes of HSF1-knockout (HSF1KO) mice with experimental cryptorchidism. StAR protein in wild-type-cryptorchid mice was transiently decreased after induction of cryptorchidism and then gradually returned to basal levels. In contrast, StAR protein in HSF1KO mice continued to decrease and failed to recover, resulting in impaired serum testosterone. StAR messenger RNA was not decreased with cryptorchidism, indicating that posttranslational modification of StAR, not its transcription, was obstructed in cryptorchidism. Other steroidogenic enzymes, including CYP11A1, 3ß-HSD, and CYP17A1, were not decreased. Lipid droplets were increased in the cytosol of HSF1KO-cryptorchid mice, suggesting dysfunctional cholesterol transportation. These findings provide insight into the role of HSF1 in Leydig cell steroidogenesis, suggesting that it maintains cholesterol transport by recovering StAR under chronic heat stress.

The HSF1-PARP13-PARP1 complex facilitates DNA repair and promotes mammary tumorigenesis.

Poly(ADP-ribose) polymerase 1 (PARP1) is involved in DNA repair, chromatin structure, and transcription. However, the mechanisms that regulate PARP1 distribution on DNA are poorly understood. Here, we show that heat shock transcription factor 1 (HSF1) recruits PARP1 through the scaffold protein PARP13. In response to DNA damage, activated and auto-poly-ADP-ribosylated PARP1 dissociates from HSF1-PARP13, and redistributes to DNA lesions and DNA damage-inducible gene loci. Histone deacetylase 1 maintains PARP1 in the ternary complex by inactivating PARP1 through deacetylation. Blocking ternary complex formation impairs redistribution of PARP1 during DNA damage, which reduces gene expression and DNA repair. Furthermore, ternary complex formation and PARP1 redistribution protect cells from DNA damage by promoting DNA repair, and support growth of BRCA1-null mammary tumors, which are sensitive to PARP inhibitors. Our findings identify HSF1 as a regulator of genome integrity and define this function as a guarding mechanism for a specific type of mammary tumorigenesis.

HSF1 and HSF3 cooperatively regulate the heat shock response in lizards.

Cells cope with temperature elevations, which cause protein misfolding, by expressing heat shock proteins (HSPs). This adaptive response is called the heat shock response (HSR), and it is regulated mainly by heat shock transcription factor (HSF). Among the four HSF family members in vertebrates, HSF1 is a master regulator of HSP expression during proteotoxic stress including heat shock in mammals, whereas HSF3 is required for the HSR in birds. To examine whether only one of the HSF family members possesses the potential to induce the HSR in vertebrate animals, we isolated cDNA clones encoding lizard and frog HSF genes. The reconstructed phylogenetic tree of vertebrate HSFs demonstrated that HSF3 in one species is unrelated with that in other species. We found that the DNA-binding activity of both HSF1 and HSF3 in lizard and frog cells was induced in response to heat shock. Unexpectedly, overexpression of lizard and frog HSF3 as well as HSF1 induced HSP70 expression in mouse cells during heat shock, indicating that the two factors have the potential to induce the HSR. Furthermore, knockdown of either HSF3 or HSF1 markedly reduced HSP70 induction in lizard cells and resistance to heat shock. These results demonstrated that HSF1 and HSF3 cooperatively regulate the HSR at least in lizards, and suggest complex mechanisms of the HSR in lizards as well as frogs.

Anti-TNF-α Agent Infliximab and Splenectomy Are Protective Against Renal Ischemia-Reperfusion Injury.

BACKGROUND: Renal ischemia-reperfusion (I/R) injury is associated with delayed graft function and results in poor long-term graft survival. We previously showed that splenectomy (SPLN) protects the kidney from I/R injury and reduces serum TNF-α levels. Herein, we further investigated the effects of SPLN on inflammatory responses and tissue injury in renal I/R by examining the expression of major inflammatory cytokines and heat shock protein 70 (HSP70). Because it was shown previously that the anti-TNF-α agent infliximab (IFX) attenuated renal I/R injury, we also investigated whether IFX administration mimics the effects of SPLN. METHODS: The left renal pedicles of adult male Wistar rats were clamped for 45 minutes and then reperfused for 24 hours; right nephrectomy and SPLN were performed immediately. A separate cohort was administered IFX 1 hour before surgery in lieu of SPLN. RESULTS: Serum creatinine and blood urea nitrogen levels were markedly elevated by I/R injury; these increases were significantly reversed by IFX. Furthermore, IFX inhibited the induction of inflammatory cytokines and HSP70 during renal I/R injury. Time-dependent profiles revealed that the expression of inflammatory cytokines was elevated immediately after I/R, whereas levels of HSP70, serum creatinine, and blood urea nitrogen began to rise 3 hours postreperfusion. Macrophages/monocytes were significantly increased in I/R-injured kidneys, but not in those administered IFX. The outcomes of SPLN mirrored those of IFX administration. CONCLUSIONS: Splenectomy and TNF-α inhibition both protect the kidney from I/R injury by reducing the accumulation of renal macrophages/monocytes and induction of major inflammatory cytokines.