The ADP-stimulated NADPH oxidase activates the ASK-1/MKK4/JNK pathway in alveolar macrophages

Abstract

The role of H2O2 as a second messenger in signal transduction pathways is well established. We show here that the NADPH oxidase-dependent production of O2*(-) and H2O2 or respiratory burst in alveolar macrophages (AM) (NR8383 cells) is required for ADP-stimulated c-Jun phosphorylation and the activation of JNK1/2, MKK4 (but not MKK7) and apoptosis signal-regulating kinase-1 (ASK1). ASK1 binds only to the reduced form of thioredoxin (Trx). ADP induced the dissociation of ASK1/Trx complex and thus resulted in ASK1 activation, as assessed by phosphorylation at Thr845, which was enhanced after treatment with aurothioglucose (ATG), an inhibitor of Trx reductase. While dissociation of the complex implies Trx oxidation, protein electrophoretic mobility shift assay detected oxidation of Trx only after bolus H2O2 but not after ADP stimulation. These results demonstrate that the ADP-stimulated respiratory burst activated the ASK1-MKK4-JNK1/c-Jun signaling pathway in AM and suggest that transient and localized oxidation of Trx by the NADPH oxidase-mediated generation of H2O2 may play a critical role in ASK1 activation and the inflammatory response.

Figure 1

ADP activates ASK1/MKK4/JNK1/2 in an H₂O₂-dependent manner. NR8383 cells were pre-incubated ± catalase (100 U/ml) for 15 min before stimulation with 400 µM ADP for the indicated times. Whole cell lysates (A–D) or ASK1 immunoprecipitates (E) were analyzed by Western blotting. ADP induced the increase in phosphorylation of JNK1/2 (A), c-Jun (B), MKK4 (C) and ASK1 (E) that was abrogated by exogenous catalase. ADP did not stimulate the phosphorylation of MKK7 (D) but TNF-α did, as previously reported. Shown are representative blots and bar graphs (B–E) represent the mean and standard deviation of photon counts obtained as described in Methods; n = 3, *p < 0.05, **p < 0.01.

Figure 2

ADP stimulation of the respiratory burst induces the dissociation of Trx from ASK1. NR8383 cells were stimulated with 400 µM ADP and ASK1 was immunoprecipitated. Catalase (100 U/ml) was added 15 min before stimulation. (A) ADP induces Trx dissociation from ASK1. (B). Catalase eliminated ADP-induced dissociation of Trx from ASK1. Experiments were repeated at least three times and the blots shown are representative of the results obtained. The bar graph (B) represents the mean and standard deviation of photon counts as described in Methods; n = 4, *p < 0.05.

Figure 3

Trx dissociation from ASK1 is enhanced by ATG. NR8383 were pretreated with 20 µM ATG for 15 min before stimulation with ADP and ASK1 was immunoprecipitated. ASK1 phosphorylation and Trx association were determined by Western blotting of the immunoprecipitates. ATG enhances ADP-mediated Trx dissociation (A) and ASK1 phosphorylation (B). The experiments were repeated three times and the blots shown are representative of the results obtained. Bar graphs represent the mean and standard deviation of photon counts obtained as described in Methods; n = 3, *p < 0.05, **p < 0.01.

Figure 4

PEMSA determination of Trx redox state. NR8383 cells were exposed to 100 µM H₂O₂ (3–4) or 400 µM ADP (5–10) for 1 or 5 min and harvested for Trx redox determination as described in Methods. Where indicated, cells were pre-incubated for 15 min with 100 U/ml catalase (7 – 8) or 2 µM DPI (9 – 10). Lane 1 represents the rat Trx alkylation ladder control. Arrows indicate the fraction of Trx in fully reduced (Trx(SH)2) or dithiol (Trx(SS)) state. PEMSA was repeated three times and the blots shown are representative of the results obtained.

Figure 5

Proposed model for activation of the JNK pathway by ADP. ADP, acting via its G-protein coupled receptor, stimulates the assembly of the NADPH oxidase (NOX2) at the plasma membrane, resulting in extracellular generation of superoxide that dismutates to hydrogen peroxide. H₂O₂ freely diffuses into cells but can be eliminated by addition of extracellular catalase. Inactive ASK1 (iASK1) is bound to reduced thioredoxin (Trx-(SH)(S⁻)), which is released from ASK1 upon its oxidation to a disulfide (Trx(S)₂) by H₂O₂, a reaction likely catalyzed by a Prx. Free ASK1 undergoes activation (aASK1) via several mechanisms including phosphorylation of Thr⁸⁴⁵, resulting in activation of the JNK module and phosphorylation of c-Jun. Reduction of oxidized Trx by NADPH and TrxR is followed by Trx re-association with ASK1, which can be delayed upon inhibition of TrxR by gold compounds such as ATG.