Citation

Nuclear Factor kappa B (NF-B) is a transcription factor family critical in the activation of pro- inflammatory responses. The NF-B pathway is regulated by oxidant-induced post-translational modifications. Protein S-glutathionylation, or the conjugation of the antioxidant molecule, glutathione to reactive cysteines inhibits the activity of inhibitory kappa B kinase beta (IKK), among other NF-B proteins. Glutathione S-transferase Pi (GSTP) is an enzyme that has been shown to catalyze protein S-glutathionylation (PSSG) under conditions of oxidative stress. The objective of the present study was to determine whether GSTP regulates NF-B signaling, S-glutathionylation of IKK, and subsequent pro-inflammatory signaling. We demonstrated that, in unstimulated cells, GSTP associated with the inhibitor of NF-B, IB. However, exposure to LPS resulted in a rapid loss of association between IB and GSTP, and instead led to a protracted association between IKK and GSTP. LPS exposure also led to increases in the S-glutathionylation of IKK. SiRNA-mediated knockdown of GSTP decreased IKK-SSG, and enhanced NF-B nuclear translocation, transcriptional activity, and pro-inflammatory cytokine production in response to lipopolysaccharide (LPS). TLK117, an isotype-selective inhibitor of GSTP, also enhanced LPS-induced NF-B transcriptional activity and pro-inflammatory cytokine production, suggesting that the catalytic activity of GSTP is important in repressing NF-B activation. Expression of both wild-type and catalytically-inactive Y7F mutant GSTP significantly attenuated LPS- or IKK-induced production of GM-CSF. These studies indicate a complex role for GSTP in modulating NF-B, which may involve S-glutathionylation of IKK proteins, and interaction with NF-B family members. Our findings suggest that targeting GSTP is a potential avenue for regulating the activity of this prominent pro-inflammatory and immunomodulatory transcription factor.