David Gius

Intracellular oxidation/reduction status in the regulation of transcription factors NF-κB and AP-1

The eukaryotic cell contains a multitude of pathways coupling environmental stimuli to the specific regulation of gene expression. Two early response transcriptional complexes, NF-kappaB and AP-1, appear to respond to environmental stressors by inducing the expression of response specific downstream genes. Both are well-characterized transcriptional regulatory factors that are induced by a wide variety of seemingly unrelated exogenous and endogenous agents and serve important roles in cell growth and differentiation, immunity, inflammation, and other preprogrammed cellular genetic processes. The activities of NF-kappaB and AP-1 are also affected following exposure to chemicals, drugs, or other agents that appear to alter the cellular oxidation/reduction (redox) status. From these observations, it has been suggested that changes in cellular oxidation/reduction status, communicated via a series of cellular redox-sensitive signaling circuitry employing metal- and thiol-containing proteins, serve as common mechanisms linking environmental stressors to adaptive cellular responses. As such, these transcription factors are ideal paradigms to study the mechanism and possible physiological significance of early response genes in the cellular response to changes in cellular redox status. In this article we summarize the evidence suggesting that cellular redox regulates these transcription factors.

Heat Shock Inhibits Radiation-induced Activation of NF-κB via Inhibition of I-κB Kinase

Radiation stimulates signaling cascades that result in the activation of several transcription factors that are believed to play a central role in protective response(s) to ionizing radiation (IR). It is also well established that heat shock alters the regulation of signaling cascades and transcription factors and is a potent radiosensitizing agent. To explore the hypothesis that heat disrupts or alters the regulation of signaling factors activated by IR, the effect of heat shock on IR-induced activation of NF-κB was determined. Irradiated HeLa cells demonstrated transient increases in NF-κB DNA binding activity and NF-κB protein nuclear localization. In addition, irradiated cells demonstrated increased I-κB phosphorylation and decreased I-κBα cytoplasmic protein levels, corresponding temporally with the increase of NF-κB DNA binding. Heat shock prior to IR inhibited the increase in NF-κB DNA binding activity, nuclear localization of NF-κB, and the phosphorylation and subsequent degradation of I-κB. I-κB kinase (IKK) immunoprecipitation assays demonstrated an increase in IKK catalytic activity in response to IR that was inhibited by pretreatment with heat. Kinetic experiments determined that heat-induced inhibition of NF-κB activation in response to IR decayed within 5 h after heating. Furthermore, pretreatment with cycloheximide, to blockde novo protein synthesis, did not alter heat shock inhibition of IR induction of NF-κB. These experiments demonstrate that heat shock transiently inhibits IR induction of NF-κB DNA binding activity by preventing IKK activation and suggests a mechanism independent of protein synthesis.

Redox Factor-1 (Ref-1) Mediates the Activation of AP-1 in HeLa and NIH 3T3 Cells in Response to Heat Shock

The early response genes, c-Fos and c-Jun, are induced by environmental stress and are thought to modulate injury processes via the induction of AP-1-dependent target genes. AP-1 activation is thought to be regulated by changes in intracellular oxidation/reduction reactions involving the redox factor-1 (Ref-1) protein. In this study, NIH 3T3 and HeLa cells were used to determine whether heat shock induces the AP-1 transcription factor via signaling pathways involving Ref-1. Reverse transcriptase-polymerase chain reaction analysis and immunoblotting demonstrated that c-Fos and c-Jun were induced 2–10 h following heat shock, and this induction was accompanied by an increase in AP-1 DNA binding. Electrophoretic mobility shift assay extracts immunodepleted of Ref-1 protein demonstrated that the increase in AP-1 DNA-binding activity following heating was dependent upon the presence of Ref-1 and that Ref-1 regulates inducible, but not basal, AP-1 DNA-binding activity. This was confirmed by the restoration of heat-inducible DNA binding upon addition of Ref-1 to immunodepleted extracts. The ability of Ref-1 from heated cells to stimulate AP-1 DNA binding was abolished by chemical oxidation and restored by chemical reduction. These results indicate that heat shock activates c-Fos/c-Jun gene expression and AP-1 DNA binding and suggests that redox-sensitive signal transduction pathways involving Ref-1 may mediate heat-induced alterations in AP-1 activation.