Heat shock protein hsp70 accelerates the recovery of heat-shocked mammalian cells through its modulation of heat shock transcription factor HSF1.

Abstract

The role of mammalian 70-kDa heat shock protein (hsp7O) in regulating cellular response to heat shock was examined by using three closely related rat cells: control Rat-1 cells, thermotolerant Rat-i (TT Rat-1) cells, and heatresistant M21 cells, a derivative of Rat-1 cells that constitutively overexpress human hsp7O. In all these cells, after a prescribed heat shock, the level of the phosphorylated form of heat shock transcription factor HSF1 and that of HSF1 The heat shock protein hsp70 appears to protect cells from thermal damage (1-3). There is also evidence that hsp7O regulates its own synthesis (4, 5). At the transcriptional level, hsp70 may be involved in modulation of the heat shock transcription factor (HSF) (6-9). HSF is a sequence-specific DNA binding protein that, in its trimeric form, binds tightly to multicopies of a highly conserved sequence motif in the promoter region of heat shock genes, termed the heat shock element (HSE).In Saccharomyces cerevisiae, HSF is a trimer bound to HSE, even in unstressed cells. Upon heat shock, HSF undergoes phosphorylation, concurrent with the transcriptional activation of heat shock genes (10,11 (8,14,(23)(24)(25). In unstressed cells, HSF1 is monomeric and non-DNA binding, probably through its interaction with hsp70. Upon heat shock, the increased level of thermally denatured or malfolded proteins may titrate the available hsp7O, setting free the hsp70-bound HSF1. The released HSF1 then assembles into a trimer, binds to HSE, and becomes phosphorylated. Transcriptional activation of heat shock genes then increases the level of hsp70. When sufficient hsp70 is made, it interacts again with activated HSF1 to form a HSF1-hsp70 complex. Finally, HSF1 dissociates from the DNA and is eventually converted to the inactive, non-DNAbinding HSF1 monomers (25). Consistent with this idea, data suggesting interaction between hsp70 and HSE-bound HSF1 have been reported (8).We have previously shown that rat cell lines that stably and constitutively express a cloned human hsp7O gene are heat resistant and have enhanced recovery of transcriptional and translational activities after heat shock (3,26). This study shows that hsp7O modulates the DNA-binding activity and phosphorylation of HSF1 after heat shock. During post-heat shock recovery, the kinetics of dephosphorylation of HSF1 and loss of the DNA-binding ability of HSF1 appear to be faster in cells overexpressing hsp7O. Treatment of cells with a phosphatase inhibitor, okadaic acid, prolongs both the phosphorylation and the DNA-binding ability of HSF1 in thermalsensitive Rat-1 cells but not in cells overexpressing human hsp70. These results support our hypothesis that hsp70 plays a role in recovery of cells after a thermal insult. MATERIALS AND METHODSCell Cultures and Heat Shock Treatment. Rat fibroblasts (Rat-1) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The transfected Rat-1 cells, constitutively expressing the intact human hsp70 (M21 and HR24) or its deletion derivatives ...

Altered Temporal Profile of Heat Shock Factor 1 Phosphorylation and Heat Shock Protein 70 Expression Induced by Heat Shock in Nucleus Tractus Solitarii of Spontaneously Hypertensive Rats

“…Overexpression of HSP70 inhibits phosphorylation of HSF1 in the nucleus at serine residues via a mechanism that involves PKC but not PKA pathway. 6,30,31 This negative feedback mechanism was proposed to explain the wellestablished reduction in HSP70 expression after repetitive exposure of cells or animals to HS. 30,32,33 It is thus conceivable that the accelerated overexpression of HSP70 triggered the same feedback regulatory mechanism, leading to our observed rapid decline in HSP70 level in the dorsomedial medulla of SHR after HS.…”

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

“…Thus, PPs appear as key linker molecules between the cellular effects of TNFa, the activation of HSF1/hsp70 stress responses and cellular susceptibility to apoptosis. Several points of evidence support this concept: (i) PPs PP1/PP2a and PP2b can be activated by TNFa, 29,39 (ii) activation of these PPs increases susceptibility to apoptotic cell death, 28,29 (iii) activation of PP1/PP2a and PP2b (calcineurin) as well as elevated intracellular calcium levels inhibit phosphorylation of HSF1 and thus activation of the HSF1/hsp70 stress response, [40][41][42] (iv) hsp70, which acts in a negative-feedback loop on HSF1 activation, 18 induces PP1/PP2a as well as PP2b and thus blocks continuing activation of HSF1 43,44 and (v) ERK and NF-kB signaling, which maintained a certain responsiveness of HSF1/hsp70 and also protected cells from apoptotic death, is a concurring mechanism to PPs, since their mutual inhibition has been repeatedly demonstrated. 45,46 In summary, the data presented provide insights into the interplay of signals essential for cell survival, stress resistance and apoptosis.…”

“…Based on the observation that hsp70 negatively regulates HSF1 18 and that extracellular hsp70 can induce TNF, 19 we hypothesized that extracellular hsp70 acts on HSF1 via TNF.…”

Invited Review: Effects of heat and cold stress on mammalian gene expression

“…In addition to positive regulation of the HSE through HSF-1, evidence also exists for negative regulation of this promoter element in mice by means of a constitutively expressed protein known as the HSE binding factor (HSE-BF) (55,66). Thus, in addition to a phosphorylation state, the ability of HSF-1 to activate transcription may also be modulated by regulatory processes that affect the binding of HSE-BF to the HSE.…”