Induction of inducible nitric oxide synthase-NO* by lipoarabinomannan of Mycobacterium tuberculosis is mediated by MEK1-ERK, MKK7-JNK, and NF-kappaB signaling pathways

Abstract

Nitric oxide (NO*) expression by inducible nitric oxide synthase (iNOS) is an important host defense mechanism against Mycobacterium tuberculosis in mononuclear phagocytes. The objective of this investigation was to examine the role of mitogen-activated protein (MAP) kinase (MAPK) and nuclear factor kappaB (NF-kappaB) signaling pathways in the regulation of iNOS and NO* by a mycobacterial cell wall lipoglycan known as mannose-capped lipoarabinomannan (ManLAM). Specific pharmacologic inhibition of the extracellular-signal-regulated kinase (ERK) or NF-kappaB pathway revealed that both these signaling cascades were required in gamma interferon (IFN-gamma)-ManLAM-induced iNOS protein and NO2- expression in mouse macrophages. Transient cotransfection of dominant-negative protein mutants of the c-Jun NH2-terminal kinase (JNK) pathway revealed that the MAP kinase kinase 7 (MKK7)-JNK cascade also mediated IFN-gamma-ManLAM induction of iNOS promoter activity whereas MKK4 did not. Overexpression of null mutant IkappaBalpha, a potent inhibitor of NF-kappaB activation, confirmed that the IkappaBalpha kinase (IKK)-NF-kappaB signaling pathway enhanced IFN-gamma-ManLAM-induced iNOS promoter activity. By contrast, activated p38mapk inhibited iNOS induction. These results indicate that combined IFN-gamma and ManLAM stimulation induced iNOS and NO. expression and that MEK1-ERK, MKK7-JNK, IKK-NF-kappaB, and p38mapk signaling pathways play important regulatory roles.

FIG. 1

IFN-γ synergizes with ManLAM in iNOS and NO₂⁻ expression. (A) RAW 264.7γNO(−) cells were stimulated with IFN-γ (10 U/ml), ManLAM (10 μg/ml), or combined IFN-γ (10 U/ml) and ManLAM (0.01 to 10 μg/ml) for 18 h, followed by the Greiss reagent assay for NO₂⁻ in the supernatant. Data shown are the means ± SD of three independent experiments. (B) Macrophages were stimulated with IFN-γ (10 U/ml), ManLAM (10 μg/ml), or both for 18 h, and nucleus-free lysates were separated by SDS-PAGE and immunoblotted with a polyclonal iNOS antibody. The immunoblot shown is representative of three independent experiments.

FIG. 2

Activation of ERK, p38^mapk, and JNK signaling pathways with ManLAM stimulation. (A) RAW 264.7γNO(−) macrophages were stimulated with 5 and 10 μg of ManLAM/ml for 30 min, followed by immunoblotting of nucleus-free whole-cell lysate with phospho-specific antibody to ERK (p-p42 and p-p44). (B) The cells were treated as for panel A, and the separated proteins were immunoblotted with phospho-specific p38^mapk antibody (p-p38). Western blots with anti-ERK1/2 (A, p44 and p42) and anti-p38^mapk (B, p38) antibodies are also shown. (C) JNK activity was determined by an in vitro kinase assay on nucleus-free lysates using c-Jun–GST as the substrate. Data shown are representative of three independent experiments.

FIG. 3

Inhibition of MEK1-ERK by PD98059 inhibits iNOS and NO₂⁻ expression by IFN-γ plus ManLAM. (A) RAW 264.7γNO(−) macrophages were pretreated with various concentrations of PD98059 (0.1 to 30 μM) or with vehicle DMSO (D) at a concentration of 0.075% for 1 h and then costimulated with IFN-γ (10 U/ml) plus ManLAM (10 μg/ml) for 18 h, followed by the Greiss reagent assay for NO₂⁻. Data shown are the means ± SD of three independent experiments. ∗∗∗, P < 0.001 (versus second bar from left). (B) Macrophages were similarly stimulated with IFN-γ plus ManLAM with or without PD98059 for 18 h, followed by immunoblotting of nucleus-free lysates with iNOS polyclonal antibody. The immunoblot shown is representative of three independent experiments.

FIG. 4

Inhibition of p38^mapk with SB203580 augmented iNOS and NO₂⁻ expression stimulated by IFN-γ plus ManLAM. (A) RAW 264.7γNO(−) macrophages were cultured with media alone, IFN-γ (10 U/ml) plus SB203580 (30 μM), IFN-γ (10 U/ml) plus ManLAM (10 μg/ml), or IFN-γ plus ManLAM plus SB203580. The cells were preincubated with SB203580 for 1 h and then cocultured with the indicated stimuli. After 18 h of stimulation, the supernatant was assayed for NO₂⁻. Data shown are the means ± SD of three independent experiments. ∗∗∗, P < 0.001 versus third bar from left. (B) Macrophages were cultured in media alone or with IFN-γ plus SB203580 or were stimulated with IFN-γ plus ManLAM with or without SB203580 for 18 h, followed by immunoblotting of nucleus-free lysate with an iNOS polyclonal antibody. The immunoblot shown is representative of three independent experiments.

FIG. 5

The effects of DN-MKK7 and DN-MKK4 on IFN-γ–ManLAM-induced iNOS-luc activity. (A) RAW 264.7γNO(−) macrophages were cotransfected with 0.3 μg of iNOS-luc plasmid and 2 μg of DN-MKK7 plasmid or 2 μg of pCMV5 empty expression vector, followed by stimulation with IFN-γ (10 U/ml) plus ManLAM (10 μg/ml). After 8 h of stimulation, the cells were lysed and the nucleus-free lysates were then measured for luciferase activity. The results are reported as fold increases in relative light units (fold RLU) and are normalized for protein concentration. Data shown are the means ± SD of three independent experiments. ∗∗, P < 0.01. (B) Macrophages were cotransfected with 0.3 μg of iNOS-luc plasmid and 2 μg of DN-MKK4 plasmid or 2 μg of LNCX empty vector, followed by IFN-γ–ManLAM costimulation. After 8 h of stimulation, the cells were lysed and the nucleus-free lysates were then measured for luciferase activity. The results are normalized for protein concentration. Data shown are the means ± SD of three independent experiments, ns, not significant.

FIG. 6

The effects of NF-κB inhibition on NO₂⁻ expression. Macrophages were pretreated with BAY 11-7082 (1 to 10 μM) or DMSO (0.02%) for 1 h and then costimulated with IFN-γ (10 U/ml) plus ManLAM (10 μg/ml) for 18 h, followed by NO₂⁻ assay. Data shown are the means ± SD of three independent experiments.

FIG. 7

The effects of a mutant (null) IκBα on IFN-γ–ManLAM-induced iNOS promoter activity. RAW 264.7γNO(−) macrophages were cotransfected with 0.3 μg of iNOS-luc plasmid and 1 μg of null IκBα plasmid or 1 μg of pCMV5 vector, followed by stimulation with IFN-γ (10 U/ml) plus ManLAM (10 μg/ml). Nucleus-free lysates were then measured for luciferase activity after 8 h of stimulation. The results are reported as fold increases in relative light units (fold RLU) and are normalized for protein concentration. Data shown are the means ± SD of three independent experiments. ∗, P < 0.05.

FIG. 8

Diagram of the proposed signaling pathways that modulate ManLAM-induced iNOS expression. The required IFN-γ costimulus pathway utilizing transcription factors IRF-1 and Stat1α is also shown.