Roles of the heat shock transcription factors in regulation of the heat shock response and beyond.

The heat shock response, characterized by increased expression of heat shock proteins (Hsps) is induced by exposure of cells and tissues to extreme conditions that cause acute or chronic stress. Hsps function as molecular chaperones in regulating cellular homeostasis and promoting survival. If the stress is too severe, a signal that leads to programmed cell death, apoptosis, is activated, thereby providing a finely tuned balance between survival and death. In addition to extracellular stimuli, several nonstressful conditions induce Hsps during normal cellular growth and development. The enhanced heat shock gene expression in response to various stimuli is regulated by heat shock transcription factors (HSFs). After the discovery of the family of HSFs (i.e., murine and human HSF1, 2, and 4 and a unique avian HSF3), the functional relevance of distinct HSFs is now emerging. HSF1, an HSF prototype, and HSF3 are responsible for heat-induced Hsp expression, whereas HSF2 is refractory to classical stressors. HSF4 is expressed in a tissue-specific manner; similar to HSF1 and HSF2, alternatively spliced isoforms add further complexity to its regulation. Recently developed powerful genetic models have provided evidence for both cooperative and specific functions of HSFs that expand beyond the heat shock response. Certain specialized functions of HSFs may even include regulation of novel target genes in response to distinct stimuli.

Genomic organization and promoter analysis of the human heat shock factor 2 gene.

Heat shock factor 2 (HSF2) is a member of the heat shock transcription factor family, which appears to be activated during differentiation and development rather than on cellular stress. Here we report the isolation and characterization of the human hsf2 gene and its 5'-flanking region. The transcription unit of the human hsf2 gene consists of 13 exons dispersed over 33 kbp of genomic DNA on chromosome 6. The hsf2 mRNA is transcribed from multiple start sites, and initiation from the major site results in a transcript of 2.45 kb. A functional promoter, as determined by the ability to direct expression of a transiently transfected luciferase reporter gene, resides in a 950-bp upstream region of the human hsf2 gene. Examination of the core promoter sequence revealed a high GC content and lack of a canonical TATA box. This feature seems to be common among various species, as comparison of the hsf2 proximal promoter sequences from human, mouse, and rat showed distinct conserved regions. Moreover, the overall architecture of the human hsf2 gene is similar to its mouse counterpart. A comparison between human hsf2 gene and other hsf genes showed striking similarities in exon size. However, the exons are assembled in an hsf-specific manner.

Disruption of heat shock factor 1 reveals an essential role in the ubiquitin proteolytic pathway.

Inhibition of proteasome-mediated protein degradation machinery is a potent stress stimulus that causes accumulation of ubiquitinated proteins and increased expression of heat shock proteins (Hsps). Hsps play pivotal roles in homeostasis and protection in a cell, through their well-recognized properties as molecular chaperones. The inducible Hsp expression is regulated by the heat shock transcription factors (HSFs). Among mammalian HSFs, HSF1 has been shown to be important for regulation of the heat-induced stress gene expression, whereas the function of HSF2 in stress response is unclear. Recent reports have suggested that both HSF1 and HSF2 are affected during down-regulation of ubiquitin-proteasome pathway (Y. Kawazoe et al., Eur. J. Biochem. 255:356-362, 1998; A. Mathew et al., Mol. Cell. Biol. 18:5091-5098, 1998; D. Kim et al., Biochem. Biophys. Res. Commun. 254:264-268, 1999). To date, however, no unambiguous evidence has been presented as to whether a single specific HSF or multiple members of the HSF family are required for transcriptional induction of heat shock genes when proteasome activity is down-regulated. Therefore, by using loss-of-function and gain-of-function strategies, we investigated the specific roles of mammalian HSFs in regulation of the ubiquitin-proteasome-mediated stress response. Here we demonstrate that HSF1, but not HSF2, is essential and sufficient for up-regulation of Hsp70 expression during down-regulation of the ubiquitin proteolytic pathway. We propose that specificity of HSF1 could be an important therapeutic target during disease pathogenesis associated with abnormal ubiquitin-dependent proteasome function.

Differentiation lineage-specific expression of human heat shock transcription factor 2.

Differentiation of multipotential hematopoietic cells into lineage-committed precursors involves the selection and maintenance of appropriate programs of gene expression, regulated by specific transcription factors. Using human K562 erythroleukemia cells capable of differentiating along erythroid and megakaryocytic lineages, we explore the differentiation-related role of heat shock transcription factor 2 (HSF2), which belongs to a family of transcription factors generally known to regulate heat shock gene expression. We demonstrate that enhanced HSF2 expression and the acquisition of HSF2 DNA binding activity are strictly specific for erythroid characteristics of K562 cells. Our results reveal a multistep regulatory process of HSF2 gene expression. In K562 cells undergoing hemin-mediated erythroid differentiation, the increase in HSF2 protein levels is preceded by transcriptional induction of the HSF2 gene, accompanied by increased HSF2 mRNA stability. In contrast, during megakaryocytic differentiation induced by the phorbol ester TPA, expression of HSF2 is rapidly down-regulated, leading to a complete loss of the HSF2 protein. These results indicate that the determination of HSF2 expression occurs at the early stages of lineage commitment. Taken together, our data suggest that HSF2 could function as a lineage-restricted transcription factor during differentiation of K562 cells along either the erythroid or the megakaryocytic pathway.

Thioredoxin is transcriptionally induced upon activation of heat shock factor 2.

Heat shock gene expression is differentially regulated in cells exposed to stress stimuli and in cells undergoing processes of differentiation and development. Regulation of the classical heat shock response is mediated by heat shock factor 1 (HSF1), whereas heat shock factor 2 (HSF2) is activated in certain differentiating cells, for example during hemin-mediated differentiation of human K562 erythroleukemia cells. Hence, the signaling pathways leading to induction of heat shock gene expression upon different stimuli are likely to be distinct. We have used RNA arbitrarily primed polymerase chain reaction to identify genes that are differentially regulated upon activation of HSF1 and HSF2. In this study, we report that thioredoxin (TRX) expression is induced in K562 cells in response to hemin in an HSF2-dependent manner. Increased TRX expression was primarily detected on the transcriptional level, subsequently leading to elevated TRX mRNA and protein levels. Hemin treatment caused no reduction in cellular glutathione concentrations, indicating that the increased TRX expression was not due to oxidative stress. Studies using cell lines where overexpression of the HSF2-beta isoform represses HSF2 activation implied that active HSF2 is required for transcriptional induction of TRX. Unlike HSF2, activation of HSF1 did not induce TRX expression. Taken together, our results suggest that HSF1 and HSF2 may regulate distinct target genes, and activation of HSF2 could be involved in the regulation of TRX expression during hemin-mediated differentiation of K562 cells.

Overexpression of HSF2-beta inhibits hemin-induced heat shock gene expression and erythroid differentiation in K562 cells.

Acquisition of heat shock factor 2 (HSF2) DNA binding activity is accompanied by induced transcription of heat shock genes in hemin-treated K562 cells undergoing erythroid differentiation. Previous studies revealed that HSF2 consists of two alternatively spliced isoforms, HSF2-alpha and HSF2-beta, whose relative abundance is developmentally regulated and varies between different tissues. To investigate whether the molar ratio of HSF2-alpha and HSF2-beta isoforms is crucial for the activation of HSF2 and whether the HSF2 isoforms play functionally distinct roles during the hemin-mediated erythroid differentiation, we generated cell clones expressing different levels of HSF2-alpha and HSF2-beta. We show that in parental K562 cells, the HSF2-alpha isoform is predominantly expressed and HSF2 can be activated upon hemin treatment. In contrast, when HSF2-beta is expressed at levels exceeding those of endogenous HSF2-alpha, the hemin-induced DNA binding activity and transcription of heat shock genes are repressed, whereas overexpression of HSF2-alpha results in an enhanced hemin response. Furthermore, the hemin-induced accumulation of globin, known as a marker of erythroid differentiation, is decreased in cells overexpressing HSF2-beta. We suggest that HSF2-beta acts as a negative regulator of HSF2 activity during hemin-mediated erythroid differentiation of K562 cells.