Identification of Xenopus heat shock transcription factor–2: conserved role of sumoylation in regulating deoxyribonucleic acid–binding activity of heat shock transcription factor–2 proteins

The exposure of 3T3 cells to a medium made hypertonic by the addition of NaCl induced activation of a heat-shock transcription factor (HSF). This activation, as monitored by gel-mobility-shift assays, occurred within 10 min of hypertonic shock and was dose-dependent in relation to the osmotic strength of the medium up to 0.7 osM. Competition analysis indicated that the effect of hypertonic shock on HSF binding activity was specific. The magnitude of the heat-shock element (HSE)-HSF binding induced by incubating the cells in a 0.7 osM medium was comparable in intensity and time course with that induced by a 44 °C heat shock. Following removal of the stressors, the decrease in HSF-HSE binding was more rapid in hypertonicity-shocked than in heat-shocked cells. Treatment of the cells with cycloheximide did not inhibit HSF-HSE binding, indicating that the activation was independent of new protein synthesis. By using a specifically directed polyclonal serum, HSF1 was identified as the transcription factor involved in the hypertonicity-induced activation. HSF was also activated when a membrane-impermeable osmolyte such as sucrose was used to increase the osmolarity of the medium. However, no HSF-HSE binding was observed after addition of glycerol (a freely membrane-permeable osmolyte) in excess. There was a temporal relationship between the hypertonicity-induced volume decrease, the increase in the intracellular K+ concentration and the induction of HSF-HSE binding. In contrast, an increase in the intracellular Na+ concentration was not required to induce HSF-HSE binding. However, unlike the heat-shock response, the activation of HSF by hypertonic shock did not lead to elongation of the RNA transcript of heat-shock protein 70.

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Rapid activation of the heat shock transcription factor, HSF1, by hypo-osmotic stress in mammalian cells

Biochemical Journal ◽

10.1042/bj3070347 ◽

1995 ◽

Vol 307 (2) ◽

pp. 347-352 ◽

Cited By ~ 18

Author(s):

L E Huang ◽

L Caruccio ◽

A Y Liu ◽

K Y Chen

Keyword(s):

Transcription Factor ◽

Heat Shock ◽

Osmotic Stress ◽

Hela Cells ◽

Mammalian Cells ◽

Environmental Changes ◽

Binding Activity ◽

Heat Shock Transcription Factor ◽

Hypotonic Medium ◽

Hsp70 Mrna

Osmoregulation is important to living organisms for survival in responding to environmental changes of water and ionic strength. We demonstrated here for the first time that exposure of HeLa cells to a hypotonic medium (30% growth medium and 70% water) prominently induced the binding activity of the heat shock transcription factor (HSF). Pretreatment of cells with cycloheximide did not inhibit the induction of HSF-binding activity, indicating that the mechanisms of induction are independent of new protein synthesis. The magnitude of hypo-osmotic stress-induced HSF-binding activity was comparable with that induced by heat shock. The induction, as monitored by gel-mobility-shift assay, occurred within 5 min of hypo-osmotic stress and persisted at least up to 4 h in HeLa cells under the hypotonic conditions. Addition of sorbitol to the hypotonic medium abolished HSF activation. Hypo-osmotic stress-induced HSF binding could also be demonstrated in HeLa cells maintained in simple sorbitol solution by decreasing the sorbitol concentration from 300 mM to 200 mM or less. Competition analysis suggests that the effects of hypo-osmotic stress on HSF-binding activity was specific. Cross-linking experiments and Western-blot analysis demonstrated that hypo-osmotic stress induced trimerization of human heat shock factor 1 (HSF1) in intact HeLa cells, suggesting that trimer formation of HSF1 was responsible for inducing HSF-binding activity in hypo-osmotically stressed cells. However, unlike heat shock response, the activation of HSF by hypo-osmotic stress did not lead to accumulation of hsp70 mRNA in HeLa cells.

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