Abstract
Induction of heat shock gene expression is mediated by specific heat shock transcription factors (HSFs), but the signaling pathways leading to activation of HSFs are poorly understood. To elucidate whether protein kinase C-responsive signaling pathways could be involved in the regulation of heat shock gene expression, we have examined the effects of the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA) on the heat-induced stress response in K562 cells. We demonstrate that TPA treatment markedly enhances heat shock gene expression during heat stress, although TPA alone does not induce the heat shock response. This TPA-mediated enhancement can initially be detected as an accelerated acquisition of DNA binding and transcriptional activity of HSF1 resulting in elevated Hsp70 protein concentrations. In the presence of TPA, the attenuation of HSF1 DNA binding activity during continuous exposure to heat shock occurs more rapidly and in concert with the appearance of newly synthesized Hsp70, which supports earlier studies on the autoregulatory role of Hsp70 in deactivation of HSF1. During heat stress, a correlation between the hyperphosphorylation of HSF1 and its transcriptional activity was observed, in both the presence and the absence of TPA. Our results show that the heat-induced stress response can be significantly modulated by activation of protein kinase C-responsive signaling pathways.
Highlights
In eukaryotic cells, the common cellular response to heat shock and other types of stress is a rapid transcriptional activation of heat shock genes resulting in increased synthesis of the heat shock proteins (Hsps).1 The initial signaling events in this well characterized physiological process, called the stress response, have not been identified
tetradecanoylphorbol 13-acetate (TPA) Treatment during Continuous Heat Shock Results in Accelerated Acquisition and Attenuation of HSF1 DNA Binding Activity—To examine the effect of TPA on the activation of HSF1 DNA binding activity, K562 cells were exposed to heat shock at 42 °C, 100 nM TPA, or to both TPA and heat shock for various time periods extending to 6 h
TPA treatment alone did not induce any heat shock element (HSE) binding activity of HSF1 (Fig. 1), TPA was obviously effective in these cells as reflected by a 2–3-fold stimulation of AP-1 DNA binding activity upon TPA treatment over a time period of 15 min to 4 h
Summary
Heat shock protein; HSF, heat shock transcription factor; HSE, heat shock element; PKC, protein kinase C; TPA, 12-O-tetradecanoylphorbol 13-acetate; 4␣-TPA, 4␣-12O-tetradecanoylphorbol 13-acetate; PAGE, polyacrylamide gel electrophoresis; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MAPK, mitogen-activated protein kinase. Treatment with arachidonate leads to HSF1 DNA binding activity, hyperphosphorylation of HSF1, and activation of hsp gene transcription [22]. Both activating and inactivating phosphorylation sites may be present on HSF1, because a study in the yeast Kluyveromyces lactis proposes that phosphorylation of HSF may serve as a regulatory mechanism to inactivate HSF [23]. 24 –26), no specific protein kinases or protein phosphatases have yet been identified to be directly involved in the regulation of HSF1 activation or in the induction of the heat shock response. To elucidate the signaling pathways involved in the regulation of stress response, we have used the phorbol ester 12-Otetradecanoylphorbol 13-acetate (TPA) to examine the effects of PKC activation on heat-induced HSF1 activation and expression of the heat shock genes hsp and hsp. The TPA-mediated effect on the heat-induced stress response shows gene specificity, because the effects on hsp and hsp gene expression are distinct
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