A High Affinity HSF-1 Binding Site in the 5′-Untranslated Region of the Murine Tumor Necrosis Factor-α Gene Is a Transcriptional Repressor

Abstract

Tumor necrosis factor-␣ (TNF␣) is a pivotal early mediator of host defenses that is essential for survival in infections. We previously reported that exposing macrophages to febrile range temperatures (FRT) (38.5-40°C) markedly attenuates TNF␣ expression by causing abrupt and premature cessation of transcription. We showed that this inhibitory effect of FRT is mediated by an alternatively activated repressor form of heat shock factor 1 (HSF-1) and that a fragment of the TNF␣ gene comprising a minimal 85-nucleotide (nt) proximal promoter and the 138-nt 5-untranslated region (UTR) was sufficient for mediating this effect. In the present study we have used an electrophoretic mobility shift assay (EMSA) to identify a high affinity binding site for HSF-1 in the 5-UTR of the TNF␣ gene and have used a chromosome immunoprecipitation assay to show that HSF-1 binds to this region of the endogenous TNF␣ gene. Mutational inactivation of this site blocks the inhibitory effect of overexpressed HSF-1 on activity of the minimal TNF␣ promoter (؊85/؉138) in Raw 264.7 murine macrophages, identifying this site as an HSF-1-dependent repressor. However, the same mutation fails to block repression of a full-length (؊1080/؉138) TNF␣ promoter construct by HSF-1 overexpression, and HSF-1 binds to upstream sequences in the regions ؊1080/ ؊845, ؊533/؊196, and ؊326/؊39 nt in EMSA, suggesting that additional HSF-1-dependent repressor elements are present upstream of the minimal ؊85-nt promoter. Furthermore, although mutation of the HSF-1 binding site in the minimal TNF␣ promoter construct abrogates HSF-1-mediated repression, the same mutation fails to abrogate repression of this construct by high levels of HSF-1 overexpression or exposure to 39.5°C. This suggests that HSF-1 might repress TNF␣ transcription through redundant mechanisms, some of which might not require high affinity binding of HSF-1. Tumor necrosis factor-␣ (TNF␣)1 is an early pivotal mediator expressed in response to infection and injury (1). Although TNF␣ is essential for optimal host defense, persistent or inappropriately high TNF␣ expression has grave consequences, including multiorgan failure and death (2-5). The pleiotropic nature of TNF␣ has lead to the evolution of stringent and redundant regulatory mechanisms imposed at transcriptional, translational, and posttranslational levels (6 -11). We reported that exposure to febrile range hyperthermia suppresses TNF␣ expression in murine peritoneal macrophages, Kupffer cells, precision-cut liver slices, the murine Raw 264.7 macrophage cell line, human monocyte-derived macrophages, and the THP1 monocyte cell line (10,(12)(13)(14)(15)(16). We showed that the predominant mechanism of suppression of TNF␣ expression is by an abrupt and early cessation of TNF␣ transcription, and that the TNF␣ gene sequence between Ϫ85 and ϩ138 is sufficient to confer temperature responsiveness in murine macrophages (15). We also showed that the heat stressactivated transcription factor, heat shock transcription factor 1 (HSF-1) is activated at febr...

Hyperthermia in the febrile range induces HSP72 expression proportional to exposure temperature but not to HSF-1 DNA-binding activity in human lung epithelial A549 cells

“…We and others have demonstrated that elements of the HS response are activated at febrile-range temperatures and regulate genes involved in inflammation and the innate immune response, including tumor necrosis factor α (Ostberg et al 2000;Singh et al 2002Singh et al , 2000, interleukin (IL)-1β (Cahill et al 1996;Fairchild et al 2000), IL-8, and granulocyte macrophage colony-stimulating factor (Rice et al 2005;Singh et al 2008), as well as HSPs. A computerassisted analysis of CXC chemokine promoters showed that almost every member of this family of neutrophil chemotaxins contains HSEs within their promoter sequences, suggesting these genes may be a newly recognized class of HSF-1-regulated genes (Nagarsekar et al 2005).…”

“…To further analyze the relationship between temperature and activation of HSF-1, we analyzed the activation of HSF-1 to its intranuclear DNA-binding state by electrophoretic mobility shift assays (EMSA) using the well-characterized HSE sequence from the human HSP72 promoter as probe (Singh et al 2002. HSF-1-binding activities in nuclear extracts prepared from A549 cells maintained at 37°C or exposed to 42°C for 1 h, 39.5°C or 41°C for 1 or 6 h, or to 38.5°C for 1, 6, or 24 h were compared (Fig.…”

TNFα mediates susceptibility to heat-induced apoptosis by protein phosphatase-mediated inhibition of the HSF1/hsp70 stress response

“…Several pieces of evidence suggest a suppressive role of HSF1/hsp70 on TNFa-triggered inflammatory responses (Figure 9b, arrow e). Thus, activation of HSF1/hsp70 inhibits NF-kB activation [31][32][33] as well as transcription of the TNFa gene 34 Figure 9 Schematic mechanism of TNFa-mediated inhibition of HSF1/hsp70 and susceptibility to apoptosis. (a) The classical pathway of TNFa-mediated apoptosis involves TNF-R1 activation and signaling through caspase-8, which subsequently activates effector caspases, such as caspase-3.…”

Toll-like Receptor Agonists and Febrile Range Hyperthermia Synergize to Induce Heat Shock Protein 70 Expression and Extracellular Release

“…Electrophoretic Mobility Shift Assay-Nuclear extracts were prepared according to the method of Schreiber et al (38) as described earlier (37,39), and total protein concentration was measured. Double-stranded oligonucleotides containing the consensus HSF1 binding sequence from the human Hsp-70 promoter 5Ј-GATCTCGGCTGGAATATTCCCGACCTG-GCAGCCGA-3Ј and the complementary strands were synthesized, annealed, and radiolabeled with [␥-32 P]ATP using T4 polynucleotide kinase (Promega) according to the manufacturer's protocol (37,39).…”

“…Double-stranded oligonucleotides containing the consensus HSF1 binding sequence from the human Hsp-70 promoter 5Ј-GATCTCGGCTGGAATATTCCCGACCTG-GCAGCCGA-3Ј and the complementary strands were synthesized, annealed, and radiolabeled with [␥-32 P]ATP using T4 polynucleotide kinase (Promega) according to the manufacturer's protocol (37,39). Twenty-microliter EMSA reactions containing 5 g of nuclear extract, 0.035 pmol of radiolabeled oligonucleotide, 1 g of poly(IC), 10 mM Tris-HCl, pH 7.8, 10% glycerol, 60 mM NaCl, 1 mM EDTA, and 1 mM dithiothreitol were incubated at room temperature for 30 min.…”