(Chest. 1999;116:91S-92S.)
© 1999
American College of Chest Physicians
Systematic Evaluation of the Mitogen-Activated Protein Kinases in the Induction of iNOS by Tumor Necrosis Factor-Alpha and Interferon-Gamma*
E. D. Chan, MD;
B. W. Winston, MD, FCCP;
S. T. Uh, MD;
L. K. Remigio and
D. W. H. Riches, PhD
*
From the Division of Pulmonary Sciences, University of Colorado Health Sciences Center and National Jewish Medical and Research Center, Denver, CO.
Correspondence to: E. D. Chan, MD, Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Health Sciences Center, National Jewish Medical and Research Center, 1400 Jackson St, Denver, CO 80262
In
mouse macrophages (M
), priming by interferon-gamma (IFN-
) is
necessary for the activation of in-ducible nitric oxide synthase
(iNOS) by tumor necrosis factor-alpha (TNF-
) or
lipopolysaccharide (LPS) and subsequent production of reactive nitrogen
species. The signal transduction pathway initiated by LPS and IFN-
in iNOS induction is well established, considered to be mediated by the
transcription factors nuclear factor-kappa B (NF-
B) and interferon
regulatory factor (IRF)-1, respectively. However, the signal
transduction pathway utilized by TNF- in the induction of iNOS is
not well characterized. Because (1) the iNOS promoter contains
cis-acting elements for activation protein (AP)-1 and
NF-B transcription factors, (2) the activation of AP-1 and NF-B
are variably activated by members of the mitogen-activated protein
kinase (MAPK) family members, and (3) the MAPKs are strongly activated
by TNF-, we examined the role of the MAPKs
(p42mapk/erk2, p46 JNK, and
p38mapk) in the induction of iNOS by TNF- and
IFN-. We first examined the effects of interleukin-4 (IL-4) on iNOS
regulation and found that IL-4 downregulated iNOS and
NO2-expression by TNF- and IFN- in murine M. We
next investigated the effects of IFN- or IL-4 on the induction of
the MAPKs by TNF- and found that whereas IFN- augmented
p42mapk/erk2 and p46 JNK activation by TNF-,
IL-4 downregulated p42mapk/erk2 and p46 JNK
activation by TNF-.
Treatment of cells with N-acetylcysteine (NAC), previously shown to
inhibit the c-Jun N-terminal kinase/stress-activated protein kinase
(JNK/SAPK) pathway between TNF receptor-associated factor-2 (TRAF2) and
MAPK/ERK kinase kinase (MEKK), revealed that both iNOS (Fig 1)
and NO2- expression were significantly
inhibited. However, NAC had no effect on IRF-1 messenger RNA expression
stimulated by IFN- and no effect on glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) transcripts (Fig 1)
. Furthermore, NAC prevented
the characteristic shape change in the macrophages when stimulated with
TNF- and IFN-. Because NAC also inhibited
p38mapk and p42mapk/erk2
activation by TNF- in addition to inhibiting p46 JNK activation, the
cells were treated with a specific MEK1 inhibitor (PD98059) and/or a
p38mapk inhibitor (SB203580), and these
inhibitors were found to have no effect on
NO2- production by TNF- and IFN-.
Preliminary transfections with 3T3 fibroblasts with the iNOS
promoter~luciferase reporter constructs and dominant-negative MEKK1
suggest that the DN-MEKK1 inhibits TNF- + IFN- induction of
iNOS (Fig 2)
.
Gel retardation assays in mouse M revealed that NAC markedly
inhibits NF-B activation and modestly suppresses AP-1 activation by
TNF-. Combined with more recent studies suggesting that MEKK and/or
JNK may enhance the activation of NF-B, we propose that
MEKK
JNKKJNK signal transduction pathway is involved in the
activation of iNOS by TNF- and IFN- by the activation of NF-B.
Speculatively, NF-B may act synergistically with IRF-1 to enhance
iNOS transcription by TNF- and
IFN-.
