Glutathione S-transferase pi modulates NF-κB activation and pro-inflammatory responses in lung epithelial cells

Abstract

Nuclear Factor kappa B (NF-κB) is a transcription factor family critical in the activation of pro- inflammatory responses. The NF-κB 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 (IKKβ), among other NF-κB 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-κB signaling, S-glutathionylation of IKK, and subsequent pro-inflammatory signaling. We demonstrated that, in unstimulated cells, GSTP associated with the inhibitor of NF-κB, IκBα. However, exposure to LPS resulted in a rapid loss of association between IκBα and GSTP, and instead led to a protracted association between IKKβ and GSTP. LPS exposure also led to increases in the S-glutathionylation of IKKβ. SiRNA-mediated knockdown of GSTP decreased IKKβ-SSG, and enhanced NF-κB 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-κB transcriptional activity and pro-inflammatory cytokine production, suggesting that the catalytic activity of GSTP is important in repressing NF-κB activation. Expression of both wild-type and catalytically-inactive Y7F mutant GSTP significantly attenuated LPS- or IKKβ-induced production of GM-CSF. These studies indicate a complex role for GSTP in modulating NF-κB, which may involve S-glutathionylation of IKK proteins, and interaction with NF-κB 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.

The role of sulfenic acids in cellular redox signaling: Reconciling chemical kinetics and molecular detection strategies

“…For example, glutaredoxins (GRX), known for their ability to deglutathionylate proteins [18, 73], can also catalyze the reverse reaction in the presence of excess GSSG, but this would again require dramatic changes in GSH:GSSG ratios to allow for this reaction to go in the reverse direction. It has similarly been suggested [26] that glutathione S -transferase π 1 (GSTP1), an enzyme that was recently found to catalyze S -glutathionylation of various target proteins [74], might do so by utilizing GSSG. GSTP has a number of domains important to its function, including a G domain is important for the activation of GSH and an H-domain important in its ligandin (protein-protein interaction) function [75].…”

Effect of biomechanical stress on endogenous antioxidant networks in bovine articular cartilage

“…This transition is largely mediated through changes in IκB phosphorylation and protein turnover . Glutathione has been shown to modulate NF‐κB signaling, both positively and negatively, through multiple mechanisms, including changes in the quantity and redox ratio of glutathione, activation of glutathione peroxidase, and by glutathionylation of IκB kinases (IKKs), IκB, or NF‐κB subunits . Our finding that 50 kPa cyclic loading increased GSH while also lowering the ratio of GSH:GSSG raises questions about how glutathione may contribute to altered NF‐κB signaling.…”

“…18 Glutathione has been shown to modulate NF-kB signaling, both positively and negatively, through multiple mechanisms, including changes in the quantity and redox ratio of glutathione, activation of glutathione peroxidase, and by glutathionylation of IkB kinases (IKKs), IkB, or NF-kB subunits. [31][32][33] Our finding that 50 kPa cyclic loading increased GSH while also lowering the ratio of GSH: GSSG raises questions about how glutathione may contribute to altered NF-kB signaling. Interestingly, the finding that 100 mM of tBHP pre-treatment reduced the acute effects of mechanical stress on cell death and proteoglycan catabolism 22 may be mediated in part through altered NF-kB signaling.…”

Protein cysteine oxidation in redox signaling: Caveats on sulfenic acid detection and quantification

“…Recently, Tew and Janssen-Heininger and their colleagues have demonstrated that glutathione S-transferase π (GSTP) can glutathionylate IKKβ with GSSG, and thereby activate NF-κB [40]. This process requires a source of GSSG, which is likely to be a GPx or Prdx6 closely associated with the target protein, so that a relatively high concentration of GSSG is present in the immediate location.…”