Dynamic redox control of NF-kappaB through glutaredoxin-regulated S-glutathionylation of inhibitory kappaB kinase beta

Abstract

The transcription factor NF-kappaB, a central regulator of immunity, is subject to regulation by redox changes. We now report that cysteine-179 of the inhibitory kappaB kinase (IKK) beta-subunit of the IKK signalosome is a central target for oxidative inactivation by means of S-glutathionylation. S-glutathionylation of IKK-beta Cys-179 is reversed by glutaredoxin (GRX), which restores kinase activity. Conversely, GRX1 knockdown sensitizes cells to oxidative inactivation of IKK-beta and dampens TNF-alpha-induced IKK and NF-kappaB activation. Primary tracheal epithelial cells from Glrx1-deficient mice display reduced NF-kappaB DNA binding, RelA nuclear translocation, and MIP-2 (macrophage inflammatory protein 2) and keratinocyte-derived chemokine production in response to LPS. Collectively, these findings demonstrate the physiological relevance of the S-glutathionylation-GRX redox module in controlling the magnitude of activation of the NF-kappaB pathway.

Fig. 1.

Inhibition of IKK activity through reversible oxidation of Cys-179 of IKK-β. (A) C10 cells transfected with WT (filled bars) or Cys-179-Ala (open bars) HA-IKK-β expression vectors were exposed to 10 ng/ml TNF-α alone or in combination with 200 μM H₂O₂ for 5 min, and IKK activity was assessed. (B) C10 cells transfected with WT (filled bars) or Cys-179-Ala (open bars) HA-IKK-β expression vectors in combination with pcDNA3 or expression vectors of Nox1 plus its coactivators, p41 and p51, were stimulated with 10 ng/ml TNF-α for 5 min before assessment of IKK-β activity. (A and B) Blots labeled “IB:HA” are Western blots for HA, and graphs show quantification by phosphoimage analysis. (C) C10 cells were treated with 200 μM H₂O₂ for 5 min, washed, and supplied with fresh media. (Top) At different time points thereafter, cells were stimulated with 10 ng/ml TNF-α for 5 min and assayed for IKK-β activity. (Middle) Western blot for IKK-γ. (Bottom) Quantification by phosphoimage analysis. (D) C10 cells were transfected with WT HA-IKK-β and exposed to 200 μM H₂O₂ for 5 min, and oxidation of IKK-β was assessed by two-dimensional gel electrophoresis. To determine the reversibility of oxidation, cells were incubated with 10 mM DTT for 30 min after treatment with H₂O₂.

Fig. 2.

S-glutathionylation of Cys-179 of IKK-β corresponds to inactivation by H₂O₂. (A) C10 cells transfected with WT or Cys-179-Ala HA-IKK-β expression vectors were exposed to 10 ng/ml TNF-α with or without 200 μM H₂O₂ for 5 min. S-glutathionylated proteins were immunoprecipitated by using an antibody against GSH, followed by detection of HA-IKK-β by Western blotting. (B) C10 cells were transfected with WT HA-IKK-β plus Nox1, p41, and p51 expression vectors, and S-glutathionylation was investigated as in A. (C) C10 cells transfected with WT HA-IKK-β expression vector were treated with 200 μM H₂O₂ for 5 min. Cells were washed, supplied with fresh media, and harvested after 20 min. S-glutathionylation of IKK-β was investigated as in A. Blots labeled “IB:HA” are control Western blots for HA.

Fig. 3.

GRX1 modulates the inhibitory effects of H₂O₂ on IKK-β and NF-κB. (A Top) Cells were treated with 200 μM H₂O₂ for 5 min, and S-glutathionylation of IKK-β was assessed as in Fig. 2A. (Middle and Bottom) Control Western blots for HA-IKK-β and HA-GRX1, respectively. (B and C) C10 cells overexpressing HA-GRX1 were exposed to agents as before and evaluated after 5 min for IKK activity (B) or after 15 min for IκB-α levels (C). The level of RelA was measured as a loading control. (D) Cells were cotransfected with 6x κB-tk-luc reporter vector and pcDNA3 (filled bars) or HA-GRX1 (open bars) expression vectors and exposed to 200 μM H₂O₂ for 5 min before treatment with 10 ng/ml TNF-α for 6 h. Luciferase units were corrected for the amount of protein and expressed as the percentage of pcDNA3-transfected, TNF-α-stimulated luciferase activity.

Fig. 4.

GRX1 knockdown dampens IKK-β and NF-κB activation. (A) C10 cells were transfected with control or GRX1 siRNA and treated with TNF-α in the presence or absence of H₂O₂ and analyzed as described before. (Upper) IKK activity. (Lower) IKK-γ Western blots. “Fold induction” shows fold increases in IKK activity over sham controls, based on phosphoimage analysis. (B) C10 cells stably transfected with 6x κB-tk-luc were transfected with control siRNA (filled bars) or siRNA for GRX1 (open bars) and treated with 200 μM H₂O₂ and 10 ng/ml TNF-α for 6 h. Luciferase units were corrected for the amount of protein and expressed as the percentage of control siRNA TNF-α-stimulated luciferase activity. (C) C10 cells were transfected with HA-IKK-β and control or GRX1 siRNA. Cells were loaded with Bio-GEE and treated with H₂O₂. Biotinylated proteins were immunoprecipitated by using a biotin antibody, followed by detection of IKK-β by Western blotting for HA. Blots labeled “IB:HA” are control Western blots for HA.

Fig. 5.

Attenuation of NF-κB activation and chemokine production by primary airway epithelial cells from Glrx1^−/− mice in response to LPS. GRX1 activity in WT cells was 37.4 units (undetectable in Glrx1^−/− cells). (A) Primary tracheal epithelial cells (MTE) were loaded with 1.5 mM Bio-GEE for 1 h, lysates were resolved by nondenaturing SDS/PAGE, and blots were reacted with streptavidin-HRP. IB:β-actin is a loading control. (B) WT or Glrx1^−/− MTE cells were treated with 1 μg/ml LPS for 4 h for evaluation of RelA nuclear translocation. Red, RelA immunoreactivity; green, nuclear Sytox green counterstain. (C) WT or Glrx1^−/− MTE cells were treated with 1 μg/ml LPS for 6 h, and NF-κB DNA binding was assessed by EMSA. NS, nonspecific binding. (D) WT or Glrx1^−/− MTE cells were treated with 1 μg/ml LPS for 24 h, and concentrations of keratinocyte-derived chemokine (KC) (filled bars) and MIP-2 (open bars) were assessed by ELISA on culture media and corrected for protein content.