Abstract
Avian cells express three HSF genes encoding a unique factor, HSF3, as well as homologues of mammalian HSF1 and HSF2. HSF1 is the major factor that mediates the heat shock signal in mammalian cells. We reported previously that cHSF3, as well as cHSF1, is activated by heat shock in chicken cells. In this study, we examined the functional differences between cHSF1 and cHSF3. Comparison of the heat-inducible DNA binding activity of cHSF1 with cHSF3 at various temperatures revealed that the latter was activated at higher temperatures than the former. At a mild heat shock, such as 41 degrees C, only cHSF1 was activated, whereas both cHSF1 and cHSF3 were activated following a severe heat shock at 45 degrees C. Heat-inducible nuclear translocation and trimerization were accompanied by DNA binding activity. We also observed that cHSF3 was activated by treating cells with higher concentrations of sodium arsenite compared to cHSF1. The DNA binding activity of cHSF3 by severe heat shock lasted for a longer period than that of cHSF1. Interestingly, the total amount of cHSF3 increased only upon severe heat shock, whereas that of HSF1 decreased. Substantial amounts of cHSF3 remained in the soluble fraction under severe heat shock, whereas cHSF1 rapidly moved to the insoluble fractions in that conditions. Comparison of transcriptional activity of the activation domains of cHSF1 and cHSF3 revealed that the activity of cHSF3 was as strong as that of cHSF1. These findings indicate that there are different thresholds for cHSF1 and cHSF3 and that cHSF3 is involved in the persistent and burst activation of stress genes upon severe stress in chicken cells. Pretreatment of cycloheximide elevated the threshold concentrations of arsenite of both factors. This suggests that denaturation of nascent polypeptides could be the first trigger for the activation of both factors, and the pathways for activation of cHSF1 and cHSF3 may be identical, or at least share some common mechanisms.
Highlights
Of proteins called heat shock proteins (HSPs)1 or stress proteins is rapidly induced
These results suggest that the DNA binding activity of cHSF1 and cHSF3 were both induced in Chicken embryo fibroblasts (CEF) by heat shock, but the threshold temperatures for their activation are different
We clearly showed that the threshold temperatures that activate HSF1 or HSF3 are different in chicken embryo fibroblasts
Summary
Cell Culture and Preparation of Whole Cell Extracts, Cytoplasmic Extracts, and Nuclear Extracts—Chicken embryo fibroblasts (CEF) were maintained in Dulbecco’s modified Eagle’s medium supplemented with 5% fetal calf serum, 2% chicken serum, and 2% tryptose phosphate broth. Gel Mobility Shift Assay—The antibody supershift experiments were performed by incubating 2.0 l of 1:10 diluted antiserum in PBS with 1–2 l of cell lysates in a total volume of 10 l. Each fraction (0.5 ml) was precipitated with trichloroacetic acid (final concentration, 10%), washed with acetone, dried, suspended in gel sample buffer, and analyzed by SDS-PAGE followed by Western blotting. The filters were blocked with 5% dry milk in PBS for 1 h at room temperature and incubated with a 1:500 dilution of rabbit antiserum against each HSF in PBS with 2% dry milk. After washing with PBS, the filters were incubated with a 1:1000 dilution of horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (Cappel) for 30 min at room temperature and washed with 0.1% Tween 20 in PBS. Transfection and Luciferase Assay—Co-transfection and luciferase assay were performed described previously [17, 29]
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