Abstract
We evaluated the effects of a transient cold shock followed by recovery and incubation at 37 degrees C on the regulation of heat shock gene expression in the IMR-90 human diploid fibroblasts and HeLa cells in tissue culture. We showed that preincubation of cells at 4 degrees C induced the synthesis and accumulation of the heat shock proteins (HSPs) upon recovery at 37 degrees C, and the degree of this induction was directly related to the time that the cells spent at 4 degrees C. Assays on the abundance of the hsp 70 transcript, the hsp 70 gene promoter activity, and the trimerization and activation of heat shock factor (HSF) to bind to its consensus heat shock element (HSE) provided evidence that this induction of the heat shock response in cells recovering from a transient cold shock is attributable to a transcriptional event mediated by the activation of HSF. Further, the induction was a response to the temperature upshift from 4 to 37 degrees C as opposed to the 4 degrees C treatment itself; quantitation of the HSE-binding activity of cells incubated at 4 degrees C without recovery and incubation at 37 degrees C gave no evidence of an activated response. Analysis of the effects of protein synthesis inhibitors demonstrated that neither cycloheximide nor puromycin was effective in blocking the induction of HSE-binding activity in cells recovering from a transient cold shock. Experiments on the time course and temperature dependence of this induction of HSE-binding activity showed that the onset, magnitude, and duration of this induction were directly proportional to the severity of the cold stress (measured by time and temperature). We discuss the possible mechanism(s) involved in this induction of the heat shock genes at 37 degrees C by a transient cold shock and the biological implications of this observation.
Highlights
We evaluated the effects of a transientcold shockfollowed by recovery and incubation at 37 "C on the regulation of heat shock gene expressionin the IMR-90 human diploidfibroblasts and HeLa cells in tissue culture
In order to better understand tmhechanism of induc- increase inHSE-binding activityof cells incubated a t 4 "C for 2 tion of heat shock proteins (HSPs) synthesis incells recovering from a transient cold h without recovery and incubation at 37 "C (Fig. 4B: compare shock, we evaluated the effects of preincubation a t 4 "C fol- lanes 1 and 5).We have evaluated the role of protein lowed by recovery and incubation a t 37 "C on the abundanceof synthesis in thiisnduction of heat shock element (HSE)-binding activity
We showed the hsp70 gene transcript,expression of a human hsp70 pro- that theinduction of HSE-binding activity uponrecovery from moter-linked reporter gene, and the HSDFNA-binding activity. a transient cold shock (2 h a t 4 "C followed by incubation at Quantitation of the hsp 70 gene transcripts by RNase protec- 37 "C for 30 min; see lane 2 of Fig. 4 B ) was not blocked by tion assay inFig. 3A showed that cold shock followed by recov- cycloheximide (60 pg/ml) addeda t 0, 2, 3, and4 h before the 4 ery andincubation of cells a t 37 "C increased the abundancoef to 37 "C temperature upshift, representedby samples inlanes the hsp70 gene transcript, and the magnitudoef the increase 7, 8, 9,and 10 of Fig. 4 B, respectively
Summary
Tein binding sites on the nitrocellulose membrane were blocked using This study of the effects of a transient cold shock on heat. The membrane was probed with a 1:200 dilution of the monoclonal antibody directed against the heat-inducible form of the HSP 72 protein. Fig. lA illustrates the pattern of protein synthesis of cells preincubated at 4 "C for 0,1,1.5,2,2.5,3,3.5,and 4h followed by recovery and incubation at 37 "C for 5 h. Specificityof the binding was preincubation of cells at 4 "C induced the synthesis of HSPs upon recovery at 37 "C, and the degree of this induction was directly related to the time that thecells spent at 4 "C A and B).We further noted that a 4-h preincubation at 4 "C followedby a 5-h recovery periodat 37 "C induced the synthesis demonstrated by competition with self, but not non-self DNA
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