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, 17 (4), 516-28

Redox Regulation of Mammalian Heat Shock Factor 1 Is Essential for Hsp Gene Activation and Protection From Stress

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Redox Regulation of Mammalian Heat Shock Factor 1 Is Essential for Hsp Gene Activation and Protection From Stress

Sang-Gun Ahn et al. Genes Dev.

Abstract

The activation of eukaryotic heat shock protein (Hsp) gene expression occurs in response to a wide variety of cellular stresses including heat shock, hydrogen peroxide, uncoupled oxidative phosphorylation, infection, and inflammation. Biochemical and genetic studies have clearly demonstrated critical roles for mammalian heat shock factor 1 (HSF1) in stress-inducible Hsp gene expression, resistance to stress-induced programmed cell death, extra-embryonic development, and other biological functions. Activation of mammalian Hsp gene expression involves the stress-inducible conversion of HSF1 from the inactive monomer to the DNA-binding competent homotrimer. Although Hsp activation is a central conserved process in biology, the precise mechanisms for stress sensing and signaling to activate HSF1, and the mechanisms by which many distinct stresses activate HSF1, are poorly understood. In this report we demonstrate that recombinant mammalian HSF1 directly senses both heat and hydrogen peroxide to assemble into a homotrimer in a reversible and redox-regulated manner. The sensing of both stresses requires two cysteine residues within the HSF1 DNA-binding domain that are engaged in redox-sensitive disulfide bonds. HSF1 derivatives in which either or both cysteines were mutated are defective in stress-inducible trimerization and DNA binding, stress-inducible nuclear translocation and Hsp gene trans-activation, and in the protection of mouse cells from stress-induced apoptosis. This redox-dependent activation of HSF1 by heat and hydrogen peroxide establishes a common mechanism in the stress activation of Hsp gene expression by mammalian HSF1.

Figures

Figure 1
Figure 1
HSF1 is reversibly activated in vitro in a redox-regulated manner. (A) Recombinant mouse HSF1 was purified as a monomer and treated by either heat shock (HS) at 42°C for 5 min or with H2O2 (200 μM at 15°C for 5 min), cross-linked with increasing concentrations of EGS (wedge) at room temperature for 20 min, resolved by SDS-PAGE, and detected by immunoblotting. The positions of protein mass standards are shown on the left, and the positions of HSF1 monomers, dimers, trimers and higher-order multimers are shown on the right. (B) Activation of HSF1 multimerization is independent of the C-terminal region. HSF1(1–290) was treated as for the full-length protein in A. (C) In vitro activation by heat shock and H2O2 is HSF1 isoform-specific. Recombinant HSF1(1–290) and HSF2(1–280), purified as described for full-length HSF1 were treated (+) with heat shock or H2O2 as described in A, followed by incubation with a 32P-labeled HSE DNA fragment and analysis by EMSAs. The arrow indicates the position of the HSF–HSE protein DNA complex. (D) HSF1 activated by heat shock (HS) or H2O2 is reversibly inactivated by reductant. Following HSF1 activation by heat shock or H2O2 as in A, DTT was added to a final concentration of 5 mM for 30 min. DTT was removed by dialysis and samples restressed (R) as in A, followed by EGS cross-linking and resolution by SDS-PAGE and immunoblotting.
Figure 2
Figure 2
HSF1 cysteine residues are required for the activation and stability of homomultimers. (A) A structural model of the mammalian HSF1 DNA-binding domain, and the positions of Cys35 and Cys105 modeled into the structural coordinates for the Kluyveromyces lactis HSF DNA-binding domain structure (Harrison et al. 1994). The HSF1 helix-turn-helix DNA-binding domain is shown in red, the loop domain is shown in yellow, and cysteines at positions 35 and 105 are shown in ball-and-stick models in yellow. This model predicts that C35 and C105 are separated by ∼20Å. (BD) Stress-induced HSF1 multimerization correlates with disulfide bond formation. The purified HSF1 wild-type, HSF1 C35S, or HSF1 C105S proteins were incubated in the absence of stress (−) or in the presence of heat shock (HS) or H2O2 (+) as in Figure 1D. DTT was added to a final concentration of 5 mM for 30 min at room temperature; samples were mixed with equal volumes of 2× SDS sample buffer with or without β-ME, followed by SDS-PAGE and immunoblotting with anti-HIS antiserum.
Figure 3
Figure 3
Stress protects HSF1 cysteine residues from modification. Wild-type HSF1 and the HSF1 C35S, HSF1 C105S, HSF1 C152S, or HSF1 V56A mutants were incubated under control (−), heat shock (HS) conditions, or in the presence of H2O2 (+) and carboxymethylated with 14C-iodoacetate, followed by SDS-PAGE and image analysis. The bottom panel shows Coomassie blue staining of the purified HSF1 used in these experiments.
Figure 4
Figure 4
Changes in HSF1 redox state in vivo as a function of heat shock and H2O2 stress. MEF hsf1−/− cells were transfected with plasmids expressing either HSF1(1–290) or HSF1(1–290) C152S, and either untreated (−) or treated with heat shock at 42°C or 500 μM H2O2 for 1 h (+). Cell lysates were incubated in the presence (+) or absence (−) of AMS, followed by SDS-PAGE and immunoblotting with anit-HSF1 antiserum. Arrows indicate the positions of oxidized (Ox) and reduced (Red) HSF1 as inferred from AMS reactivity.
Figure 5
Figure 5
Redox regulation is essential for HSF1 activation in vivo. (A) hsf1−/− MEFs were used to generate stable cell lines harboring the empty vector (V) or expressing wild-type (WT) or the indicated mutants of HSF1. Immunoblotting, using actin levels as a loading control, demonstrates that the cell lines selected express equivalent levels of HSF1 protein. (B,C) The stress-inducible DNA-binding activity from cell lines expressing wild-type and the indicated mutant forms of HSF1 was assayed by EMSA using a 32P-labeled HSE DNA fragment from the Hsp70 promoter. Cell cultures were treated at 37°C for control conditions (−) or with stress conditions (+) via 500 μM H2O2 at 37°C (B) or heat shock at 42°C for 1 h (C). The arrows indicate the positions of the free HSE DNA fragment and the HSF1–HSE protein DNA complex.
Figure 6
Figure 6
Redox regulation is essential for HSF1 stress-induced nuclear translocation. (A) Mouse MEF hsf1−/− cells were transiently transfected with the wild-type or indicated mutant alleles of HSF1 that were Flag-epitope-tagged. After 48 h, cells were left untreated (Control), heat shocked at 42°C (Heat shock), or treated with 500 μM H2O2 for 1 h. HSF1 was detected by indirect immunofluorescence with anti-Flag antibody and FITC-conjugated secondary antibody, with nuclear DNA staining with DAPI, and cell visualization with Nomarski optics. Shown are representative cells from two independent experiments. (B) Subcellular fractionation of HSF1 and the HSF1 C35,105S proteins. hsf1−/− MEF cells were transiently transfected with either empty vector (V) or vectors expressing wild-type (WT) or the C35,105S HSF1 mutant. Cells were treated under control (−), heat shock, or H2O2 (+) conditions, fractionated into nuclear and cytosolic components, and resolved by SDS-PAGE and HSF1-Flag; actin and c-fos were detected by immunoblotting with ant-Flag, anti-actin, and anti-c-fos antibody as described in Materials and Methods.
Figure 6
Figure 6
Redox regulation is essential for HSF1 stress-induced nuclear translocation. (A) Mouse MEF hsf1−/− cells were transiently transfected with the wild-type or indicated mutant alleles of HSF1 that were Flag-epitope-tagged. After 48 h, cells were left untreated (Control), heat shocked at 42°C (Heat shock), or treated with 500 μM H2O2 for 1 h. HSF1 was detected by indirect immunofluorescence with anti-Flag antibody and FITC-conjugated secondary antibody, with nuclear DNA staining with DAPI, and cell visualization with Nomarski optics. Shown are representative cells from two independent experiments. (B) Subcellular fractionation of HSF1 and the HSF1 C35,105S proteins. hsf1−/− MEF cells were transiently transfected with either empty vector (V) or vectors expressing wild-type (WT) or the C35,105S HSF1 mutant. Cells were treated under control (−), heat shock, or H2O2 (+) conditions, fractionated into nuclear and cytosolic components, and resolved by SDS-PAGE and HSF1-Flag; actin and c-fos were detected by immunoblotting with ant-Flag, anti-actin, and anti-c-fos antibody as described in Materials and Methods.
Figure 7
Figure 7
HSF1 redox activation is required for Hsp induction in response to heat shock and H2O2 stress. hsf−/− MEF stable cells lines with either vector or expressing the wild-type and indicated HSF1 alleles were treated under control (37°C), heat shock (42°C for 1 h; left panel), or H2O2 (500 μM for 24 h; right panel) conditions. After a 24-h recovery, total cell extracts were prepared, then fractionated by SDS-PAGE, and Hsp70, Hsp27, and actin were detected by immunoblotting with the respective protein-specific antibody.
Figure 8
Figure 8
HSF1 redox activation protects cells from stress-induced apoptosis. Hsf1−/− stable cell lines harboring the empty vector (V), wild-type HSF1 (WT), or the indicated mutant derivatives were incubated under control conditions (37°C), exposed to heat shock (44°C for 1 h followed by 6 h at 37°C), or 500 μM H2O2 at 37°C for 6 h. Relative apoptosis of these cultures was determined as described in Materials and Methods and plotted on the y axis as the Relative Apoptosis index.

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