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Organisms must be able to sense and respond rapidly to changes in their environment in order to maintain homeostasis and survive. Induction of heat shock proteins (Hsps) is a common cellular defense mechanism for promoting survival in response to various stress stimuli. Heat shock factors (HSFs) are transcriptional regulators of Hsps, which function as molecular chaperones in protecting cells against proteotoxic damage. Mammals have three different HSFs that have been considered functionally distinct: HSF1 is essential for the heat shock response and is also required for developmental processes, whereas HSF2 and HSF4 are important for differentiation and development. Specifically, HSF2 is involved in corticogenesis and spermatogenesis, and HSF4 is needed for maintenance of sensory organs, such as the lens and the olfactory epithelium. Recent evidence, however, suggests a functional interplay between HSF1 and HSF2 in the regulation of Hsp expression under stress conditions. In lens formation, HSF1 and HSF4 have been shown to have opposite effects on gene expression. In this chapter, we present the different roles of the mammalian HSFs as regulators of cellular stress and developmental processes. We highlight the interaction between different HSFs and discuss the discoveries of novel target genes in addition to the classical Hsps.

Objective: To investigate the cause of the enhanced intracellular replication of Salmonella enteritidis in HLA-B27-transfected U937 human monocytic cells and the contribution of HLA-B27 heavy chain (HC) misfolding., Methods: U937 monocytic cell transfectants stably expressing pSV2neo resistant vector (mock), wild-type HLA-B27, or mutated HLA-B27 HCs with amino acid substitutions in the B pocket were differentiated, infected with S enteritidis, and treated with signaling pathway inhibitors or specific p38 small interfering RNA (siRNA). The numbers of living intracellular bacteria were determined with the colony-forming unit method. To visualize S enteritidis, the bacteria were transformed with green fluorescent protein, and studied by microscopy., Results: Treatment with the p38 MAPK inhibitors or with p38 siRNA enhanced the replication of S enteritidis in U937 transfectants, whereas the other inhibitors had no effect. In mock-transfected cells and in cells expressing the mutated B27 HCs in which the misfolding had been corrected, p38 inhibitors impaired their ability to resist the replication of bacteria (mock, B27.A2B, B27.E45M, and B27.C67A). In contrast, the number of intracellular bacteria was not significantly increased in p38 inhibitor-treated cells expressing misfolded B27 HCs (B27g, B27cDNA, and B27.H9F)., Conclusion: Our results show that p38 activity plays a crucial role in controlling intracellular S enteritidis in U937 cells. Enhanced replication of bacteria in B27-expressing cells requires that the HCs contain glutamic acid at position 45 and cysteine at position 67. Furthermore, in transfectants expressing misfolded B27 HCs, p38 inhibition had no significant effect on bacterial replication, suggesting that in these cells, the p38 pathway may not function properly.

Targeting of the Hsp function in tumor cells is currently being assessed as potential anticancer therapy. An improved understanding of the molecular signals that trigger or attenuate the stress protein response is essential for advances to be made in this field. The present study provides evidence that the membrane fluidizer benzyl alcohol (BA), a documented nondenaturant, acts as a chaperone inducer in B16(F10) melanoma cells. It is demonstrated that this effect relies basically on heat shock transcription factor 1 (HSF1) activation. Under the conditions tested, the BA-induced Hsp response involves the up-regulation of a subset of hsp genes. It is shown that the same level of membrane fluidization (estimated in the core membrane region) attained with the closely analogous phenethyl alcohol (PhA) does not generate a stress protein signal. BA, at a concentration that activates heat shock genes, exerts a profound effect on the melting of raft-like cholesterol-sphingomyelin domains in vitro, whereas PhA, at a concentration equipotent with BA in membrane fluidization, has no such effect. Furthermore, through the in vivo labeling of melanoma cells with a fluorescein labeled probe that inserts into the cholesterol-rich membrane domains [fluorescein ester of polyethylene glycol-derivatized cholesterol (fPEG-Chol)], we found that, similarly to heat stress per se, BA, but not PhA, initiates profound alterations in the plasma membrane microdomain structure. We suggest that, apart from membrane hyperfluidization in the deep hydrophobic region, a distinct reorganization of cholesterol-rich microdomains may also be required for the generation and transmission of stress signals to activate hsp genes.

The heat shock response is a defense reaction activated by proteotoxic damage induced by physiological or environmental stress. Cells respond to the proteotoxic damage by elevated expression of heat shock proteins (Hsps) that function as molecular chaperones and maintain the vital homeostasis of protein folds. Heat shock factors (HSFs) are the main transcriptional regulators of the stress-induced expression of hsp genes. Mammalian HSF1 was originally identified as the transcriptional regulator of the heat shock response, whereas HSF2 has not been implicated a role in the stress response. Previously, we and others have demonstrated that HSF1 and HSF2 interact through their trimerization domains, but the functional consequence of this interaction remained unclear. We have now demonstrated on chromatin that both HSF1 and HSF2 were able to bind the hsp70 promoter not only in response to heat shock but also during hemin-induced differentiation of K562 erythroleukemia cells. In both cases an intact HSF1 was required in order to reach maximal levels of promoter occupancy, suggesting that HSF1 influences the DNA binding activity of HSF2. The functional consequence of the HSF1-HSF2 interplay was demonstrated by real-time reverse transcription-PCR analyses, which showed that HSF2 was able to modulate the HSF1-mediated expression of major hsp genes. Our results reveal, contrary to the predominant model, that HSF2 indeed participates in the transcriptional regulation of the heat shock response.

The transition from normal growth conditions to stressful conditions is accompanied by a robust upregulation of heat shock proteins, which dampen the cytotoxicity caused by misfolded and denatured proteins. The most prominent part of this transition occurs on the transcriptional level. In mammals, protein-damaging stress leads to the activation of heat shock factor 1 (HSF1), which binds to upstream regulatory sequences in the promoters of heat shock genes. The activation of HSF1 proceeds through a multi-step pathway, involving a monomer-to-trimer transition, nuclear accumulation and extensive posttranslational modifications. In addition to its established role as the main regulator of heat shock genes, new data link HSF 1 to developmental pathways. In this chapter, we examine the established stress-related functions and prospect the intriguing role of HSF 1 as a developmental coordinator.

Heat-shock factors (HSFs) are associated with multiple developmental processes, but their mechanisms of action in these processes remain largely enigmatic. Hsf2-null mice display gametogenesis defects and brain abnormalities characterized by enlarged ventricles. Here, we show that Hsf2-/- cerebral cortex displays mispositioning of neurons of superficial layers. HSF2 deficiency resulted in a reduced number of radial glia fibers, the architectural guides for migrating neurons, and of Cajal-Retzius cells, which secrete the positioning signal Reelin. Therefore, we focused on the radial migration signaling pathways. The levels of Reelin and Dab1 tyrosine phosphorylation were reduced, suggesting that the Reelin cascade is affected in Hsf2-/- cortices. The expression of p35, an activator of cyclin-dependent kinase 5 (Cdk5), essential for radial migration, was dependent on the amount of HSF2 in gain- and loss-of-function systems. p39, another Cdk5 activator, displayed reduced mRNA levels in Hsf2-/- cortices, which, together with the lowered p35 levels, decreased Cdk5 activity. We demonstrate in vivo binding of HSF2 to the p35 promoter and thereby identify p35 as the first target gene for HSF2 in cortical development. In conclusion, HSF2 affects cellular populations that assist in radial migration and directly regulates the expression of p35, a crucial actor of radial neuronal migration.

SUMO (small ubiquitin-like modifier) modification regulates many cellular processes, including transcription. Although sumoylation often occurs on specific lysines within the consensus tetrapeptide PsiKxE, other modifications, such as phosphorylation, may regulate the sumoylation of a substrate. We have discovered PDSM (phosphorylation-dependent sumoylation motif), composed of a SUMO consensus site and an adjacent proline-directed phosphorylation site (PsiKxExxSP). The highly conserved motif regulates phosphorylation-dependent sumoylation of multiple substrates, such as heat-shock factors (HSFs), GATA-1, and myocyte enhancer factor 2. In fact, the majority of the PDSM-containing proteins are transcriptional regulators. Within the HSF family, PDSM is conserved between two functionally distinct members, HSF1 and HSF4b, whose transactivation capacities are repressed through the phosphorylation-dependent sumoylation. As the first recurrent sumoylation determinant beyond the consensus tetrapeptide, the PDSM provides a valuable tool in predicting new SUMO substrates.

Engagement of death receptors induces caspase activation and apoptosis. A recent study reported altered protein expression, including increased Hsp70 levels during CD95-mediated apoptosis. Here, we examined the mechanism underlying increased Hsp70 levels in cells challenged with a monoclonal antibody directed against the CD95 receptor. Levels of Hsp70 were found to increase in a dose-dependent manner, occurring independently of either heat shock factor 1 activation or the accumulation of Hsp70 messenger ribonucleic acid (mRNA), suggesting the involvement of posttranslational modifications. Inhibition of translation and de novo protein synthesis by cycloheximide resulted in Hsp70 protein levels diminishing over time in control cells, whereas its level remained constant during CD95 signaling. In addition, death receptor activation through exposure of cells to tumor necrosis factor-related apoptosis-inducing ligand did not alter Hsp70 levels. These findings demonstrate that receptor-specific signaling through the CD95 increases the stability of Hsp70 protein, rather than mRNA, when compared with control cells. The results describe a novel mechanism of heat shock protein accumulation, where increased protein stability and reduced turnover, is the mechanism by which Hsp70 accumulates in cells during CD95-mediated apoptosis.

Objective: To reveal the cause of the impaired elimination of Salmonella enteritidis in HLA-B27-transfected human monocytic cells and to study whether the B pocket of HLA-B27 contributes to these modulatory effects., Methods: Stable U937 cell transfectants expressing HLA-A2, B27, or different forms of B27 with amino acid substitutions in the B pocket were prepared. Mock-transfected cells were prepared using the antibiotic resistance vector (pSV2neo) alone. Cells were differentiated, infected with S enteritidis, and the number of live intracellular S enteritidis organisms was determined using the colony-forming unit method. To visualize intracellular S enteritidis, the bacteria were transformed with green fluorescent protein (GFP), and studied by confocal microscopy., Results: Cells expressing wild-type HLA-B27 were more permissive of intracellular replication of S enteritidis compared with mock-transfected or A2-transfected controls. Cells expressing B27 with an altered B pocket composition having either 6 amino acid substitutions (B27.A2B; substitutions H9F, T24A, E45M, I66K, C67V, and K70H) or a single substitution (B27.E45M) were no longer permissive of S enteritidis replication. In contrast, cells expressing B27 with the single substitution of F for H at position 9 (B27.H9F) retained their permissiveness. Studies using GFP-transformed S enteritidis confirmed that the increase in the amount of intracellular bacteria in B27-expressing cells was due to replication of the bacteria., Conclusion: Our data indicate that HLA-B27 expression modulates the host-microbe interaction that results in an impaired capacity of monocytes to resist intracellular replication of S enteritidis. The phenotype is dependent on glutamic acid at position 45 in the B pocket and, thus, may be due to properties of the B27 heavy chain that are related to this residue. The ability of HLA-B27 to confer susceptibility to Salmonella-triggered reactive arthritis may occur, at least in part, through these modulatory effects.

Nuclear stress granules are subnuclear compartments that form in response to heat shock and other stress stimuli. Although many components of nuclear stress granules have been identified, including HSF1 and pre-mRNA processing factors, their function remains a mystery. A paper in this issue describes the stress-induced transcriptional activation of one of the nuclear stress granule target sites, a heterochromatic region that has been considered silent (Jolly et al., 2004). These intriguing findings will certainly give the research of these structures a new twist.

Objective: To study whether HLA-B27 modifies some key factors controlling inflammatory responses on lipopolysaccharide (LPS) stimulation in human monocytic cells., Methods: U937 human monocytic cells were stably transfected with either HLA-B27 genomic DNA, HLA-B27 complementary DNA, HLA-A2 genomic DNA, or with the resistant vector pSV2neo (mock) alone. The cells were stimulated with LPS. Electrophoretic mobility shift assay was performed to determine nuclear factor kappaB (NF-kappaB) and heat-shock factor 1 activities, Western blotting was performed to detect the expressions of inhibitory kappaBalpha (IkappaBalpha) and heat-shock proteins (HSPs), and enzyme-linked immunosorbent assay was performed to measure tumor necrosis factor alpha (TNFalpha) secretion., Results: The expression of HLA-B27 modulated the response to LPS in U937 human monocytic cells. Stimulation with LPS led to faster degradation of IkappaBalpha regulatory proteins, accompanied by faster and prolonged activation of NF-kappaB in HLA-B27-expressing cells compared with HLA-A2 and mock transfectants. The secretion of TNFalpha upon LPS stimulation correlated well with the activation of NF-kappaB. No activation of the heat-shock response was observed., Conclusion: Our data indicate that HLA-B27 has effects on host responses to LPS that are unrelated to antigen presentation. Two crucial events in the development of arthritis, the activation of NF-kappaB and the secretion of TNFalpha, were found to be enhanced in HLA-B27-expressing cells upon LPS stimulation. Because LPS is known to be present in the inflamed joints of patients with reactive arthritis (ReA), the enhanced inflammatory response of HLA-B27-positive cells upon LPS stimulation offers an attractive explanation for the role of HLA-B27 in the development of ReA.