IkappaB is a sensitive target for oxidation by cell-permeable chloramines: inhibition of NF-kappaB activity by glycine chloramine through methionine oxidation

Abstract

Hypochlorous acid (HOCl) is produced by the neutrophil enzyme, myeloperoxidase, and reacts with amines to generate chloramines. These oxidants react readily with thiols and methionine and can affect cell-regulatory pathways. In the present study, we have investigated the ability of HOCl, glycine chloramine (Gly-Cl) and taurine chloramine (Tau-Cl) to oxidize IkappaBalpha, the inhibitor of NF-kappaB (nuclear factor kappaB), and to prevent activation of the NF-kappaB pathway in Jurkat cells. Glycine chloramine (Gly-Cl) and HOCl were permeable to the cells as determined by oxidation of intracellular GSH and inactivation of glyceraldehyde-3-phosphate dehydrogenase, whereas Tau-Cl showed no detectable cell permeability. Both Gly-Cl (20-200 muM) and HOCl (50 microM) caused oxidation of IkappaBalpha methionine, measured by a shift in electrophoretic mobility, when added to the cells in Hanks buffer. In contrast, a high concentration of Tau-Cl (1 mM) in Hanks buffer had no effect. However, Tau-Cl in full medium did modify IkappaBalpha. This we attribute to chlorine exchange with other amines in the medium to form more permeable chloramines. Oxidation by Gly-Cl prevented IkappaBalpha degradation in cells treated with TNFalpha (tumour necrosis factor alpha) and inhibited nuclear translocation of NF-kappaB. IkappaBalpha modification was reversed by methionine sulphoxide reductase, with both A and B forms required for complete reduction. Oxidized IkappaBalpha persisted intracellularly for up to 6 h. Reversion occurred in the presence of cycloheximide, but was prevented if thioredoxin reductase was inhibited, suggesting that it was due to endogenous methionine sulphoxide reductase activity. These results show that cell-permeable chloramines, either directly or when formed in medium, could regulate NF-kappaB activation via reversible IkappaBalpha oxidation.

Figure 1. (A) GAPDH inactivation and (B) GSH loss in Jurkat cells treated with Tau-Cl (▼) Gly-Cl (●) or HOCl (○) in HBSS for 15 min

To stop the reaction, methionine was added to scavenge any unreacted oxidant. Cytoplasmic extracts were assayed for GAPDH activity or GSH as described in the Materials and methods section. Results are means±S.D. for at least three experiments. The GSH concentration in the control cells was 5.9 nmol.

Figure 2. Concentration-dependent oxidation of IκBα in Jurkat cells treated in HBSS

Concentration-dependent oxidation of IκBα in Jurkat cells treated in HBSS with (A) Gly-Cl (C) HOCl or (D) Tau-Cl, assessed after 15 min, the lower panel shows actin as a loading control; (B) time course of IκBα oxidation by 100 μM Gly-Cl; (E) concentration dependence for cells that were lysed before treating with Tau-Cl for 10 min. All reactions were stopped by the addition of 1 mM methionine. All cell lysates were assessed for oxidation of IκBα by running samples by SDS/15% PAGE for 2.5 h and blotting for IκBα, as described in the Materials and methods section. Oxidized IκB (ox IκB) is evident as a slower migrating band.

Figure 3. Concentration-dependent oxidation of IκBα in Jurkat cells treated in complete medium with (A) Tau-Cl (B) Gly-Cl or (C) HOCl for 30 min

Analyses were performed as in Figure 2 with the lower panel showing actin as a loading control. ox IκB, oxidized IκB.

Figure 4. Reversal of IκBα oxidation by Msr

Cells were treated with 150 μM Gly-Cl for 15 min. Lysates from Gly-Cl-treated cells containing 20 mM DTT were treated with 5 μg of MsrA, 5 μg of MsrB or both enzymes for 15 min at 37 °C, or with 10 units of AP for 30 min. Blots were analysed with anti-IκBα antibodies as in Figure 2. Similar results were obtained from three independent experiments. ox IκB, oxidized IκB.

Figure 5. Intracellular persistence of oxidized IκBα

Jurkat cells were pre-treated with 150 μM Gly-Cl and, after quenching the chloramine by the addition of 1 mM methionine, were returned to complete medium for the indicated times. Cell lysates were assessed for oxidation of IκBα as in Figure 2. (A) Time course for recovery of IκBα. (B) Effects of pre-treatment with 20 μM cycloheximide (CHX) for 1 h. ox IκB, oxidized IκB. (C) Effects of pre-treatment with 30 μM DNCB for 10 min. The histogram shows relative band densities of cells treated or not with DNCB from four independent experiments.

Figure 6. Effects of Gly-Cl and Tau-Cl on the response of IκBα to TNFα

Jurkat cells were pre-treated with 150 μM Tau-Cl or Gly-Cl for 15 min in HBSS then stimulated with 20 ng/ml TNFα for 30 min. Analyses were performed as in Figure 2. Band intensities were measured by densitometry and normalized to the untreated control, which was set to 100%. Results are means±S.D. for four experiments *P<0.001, compared with the untreated cells; all other treatments were not statistically significant.

Figure 7. Suppression of TNFα-induced nuclear transfer of NF-κB by Gly-Cl

Cells were treated with either Gly-Cl or Tau-Cl, as indicated, for 15 min at 37 °C. Fresh medium containing TNFα (20 ng/ml) (lanes 1–4) or medium alone (lanes 5 and 6) was added and cells were incubated for a further 45 min. Nuclear extracts were prepared for EMSA. Lanes 2, 3 and 4 were all from TNFα-stimulated cells. Lane 3, pre-treated with Gly-Cl. Lane 4, pre-treated with Tau-Cl. Anti-p65 was added to the extract in lane 1, where ss p65 indicates the supershifted p65 subunit of NF-κB, and ns indicates non-specific bands. Lane 7 represents control cells. Similar results were obtained in three independent experiments.

Figure 8. Effects of Gly-Cl and Tau-Cl on TNFα-mediated translocation of NF-κB to the nucleus in Jurkat cells

After 30 min of treatment with 100 μM Gly-Cl or 1 mM Tau-Cl, cells were stimulated with TNFα (20 ng/ml) for 30 min and analysed for translocation of NF-κB to the nucleus with anti-p65 antibody. (B, D, F and H) show cells that were counterstained with Hoechst 33342 to visualize the nuclei. (A, B) Jurkat cells without TNFα-stimulation. (C, D) TNFα-stimulated Jurkat cells. (E, F) TNFα-stimulated cells after pre-treatment with Tau-Cl. (G, H) TNFα-stimulated cells after pre-treatment with Gly-Cl. In (D) and (F), but not (B) and (H), there is co-localization of the fluorescence-conjugated antibody and nuclear stain.