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The zinc finger protein A20 is encoded by an immediate early response gene and acts as an inhibitor of nuclear factor (NF)-kappaB-dependent gene expression induced by different stimuli, including tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta). Toll-like receptor 2 (TLR2) and TLR4 have been found to transduce, respectively, peptidoglycan (PGN) and lipopolysaccharide (LPS) signals for the activation of NF-kappaB and the production of inflammatory cytokines. Here, we have examined the role of A20 in TLR-mediated NF-kappaB-dependent gene expression in human airway epithelial cells (AECs). Stimulation with LPS and PGN resulted in a significant increase in the level of A20 mRNA in primary cultured AECs and in NCI-H292 AECs. LPS and PGN induced activation of the IL-8 promoter both in NCI-H292 AECs and in HEK293 cells expressing either TLR2 or TLR4 plus MD-2. Dominant-negative myeloid differentiation protein and a mutant form of IkappaBalpha attenuated this PGN- or LPS-induced activation of the IL-8 promoter. Furthermore, overexpression of A20 inhibited activation of both NF-kappaB and the IL-8 promoter by PGN or LPS in these cells. Taken together, our results suggest that A20 may function as a negative regulator of TLR-mediated inflammatory responses in the airway, thereby protecting the host against harmful overresponses to pathogens.

Exhaled nitric oxide (NO) is increased in individuals with bronchial asthma. NO may have antiinflammatory and proinflammatory effects; however, its role in bronchial asthma is unclear. In the present study, to clarify this issue we examined the effect of NO in inducing activator protein-1 (AP-1) activation in human bronchial epithelial cells (BEC) and a role of apoptosis signal-regulating kinase1 (ASK1), an upstream kinase kinase of c-Jun-NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) in NO-mediated AP-1 activation. The results showed that (1) the reactive nitrogen generating species NOR-1(+/--(E)-methyl-2-[(E)-hydroxykmino]-5-nitro-6-methoxy-3-hexeneamide]) induced AP-1 activation determined by AP-1-dependent luciferase gene activity, and an NO scavenger, carboxyl-PTIO, attenuated NOR-1-induced AP-1 activation; (2) NOR-1 phosphorylated ASK1, JNK, and p38 MAPK; and (3) transient transfection of the dominant negative form of AKS1 attenuated NOR-1-induced AP-1 activation in BEC. To further characterize the role of ASK-1 cascade, the dominant negative form of ASK1-stable transfected porcine artery endothelial (PAE) cells were used. AP-1 activity and JNK and p38 MAPK phosphorylation were depressed in the dominant-negative form of ASK1-stable transfected PAE cells. These results indicate that NO is capable of inducing AP-1 activation, and that ASK1-p38 MAPK/JNK cascade regulates AP-1 activation in NO-stimulated BEC.

Myofibroblasts play an important role in the fibrogenic process of pulmonary fibrosis. Transforming growth factor (TGF)-beta is well known to induce the phenotypic modulation of fibroblasts to myofibroblasts; however, the intracellular signal regulating induction of the myofibroblastic phenotype of human lung fibroblasts (HLF) has not been determined. In the present study, we examined the role of the mitogen-activated protein kinase (MAPK) superfamily in inducing the phenotypic modulation of HLF to myofibroblasts characterized by alpha-smooth-muscle actin expression, in order to clarify this issue. The results showed that: (1) TGF-beta1 caused the phenotypic modulation of HLF to myofibroblasts in a dose- and a time-dependent manner; (2) TGF-beta1 induced increases in c-Jun-NH2- terminal kinase (JNK), p38 MAPK, and extracellular signal-regulated kinase (Erk) phosphorylation and activity; (3) the inhibitors CEP-1347, SB 203580, and PD 98059 attenuated TGF-beta1-induced JNK, p38 MAPK, and Erk activity, respectively; and (4) CEP-1347, but not SB 203580 or PD 98059, attenuated the TGF-beta1-induced phenotypic modulation of HLF to myofibroblasts in a dose-dependent manner. These results indicate that TGF-beta1 is capable of inducing the myofibroblastic phenotype of HLF, and that JNK regulates the phenotypic modulation of TGF-beta1-stimulated HLF to myofibroblasts.

Airway smooth muscle (ASM) cells, which have been regarded as having contractile properties in response to contractile inflammatory mediators, may also participate in airway inflammatory response by expressing various cytokines, including RANTES. However, the intracellular signal that regulates cytokine expression in ASM cells has not been determined. In the present study, we examined the role of p38 mitogen-activated protein (MAP) kinase and extracellular signal-regulated kinase (Erk) in RANTES production by ASM cells stimulated by platelet-activating factor (PAF) and tumor necrosis factor (TNF)-alpha. The results showed that PAF induced the threonine and tyrosine phosphorylation of p38 MAP kinase and Erk, and p38 MAP kinase and Erk activity. SB 203580 and PD 98059 almost completely inhibited p38 MAP kinase and Erk activity, respectively. SB 203580 and PD 98059 partially inhibited and acted additively to inhibit PAF-induced RANTES production. PAF also induced c-Jun-NH(2)-terminal kinase ( JNK) phosphorylation. TNF-alpha induced p38 MAP kinase and Erk phosphorylation, but neither SB 203580 nor PD 98059 inhibited RANTES production. These results indicate that both p38 MAP kinase and Erk involve RANTES production by ASM cells stimulated with PAF, but not TNF-alpha, and that the role of p38 MAP kinase and Erk in RANTES production by ASM cells appears to be stimulus-dependent.

Air pollutants including diesel exhaust particles (DEPs) have been shown to enhance allergic responses. DEPs stimulate airway epithelial cells to produce various cytokines; however, the intracellular signal transduction pathway and the involvement of reduction and oxidation (redox) control in DEP-activated signaling have not been determined. In the present study, we therefore examined the role of p38 mitogen-activated protein (MAP) kinase in DEP-induced interleukin 8 (IL-8) and RANTES production by human bronchial epithelial cells (BECs) in order to clarify the intracellular signal transduction pathway that regulates IL-8 and RANTES production. In addition, we also examined the effect of a thiol-reducing agent, N-acetylcysteine (NAC), on DEP-induced p38 MAP kinase activation and cytokine production in order to clarify the redox control mechanism in DEP-induced p38 MAP kinase activation and IL-8 and RANTES production. The results showed that DEP induced IL-8 and RANTES production and the threonine and tyrosine phosphorylation of p38 MAP kinase, reflecting the activation of p38 MAP kinase in BECs. SB 203580, as the specific inhibitor of p38 MAP kinase activity, inhibited DEP-induced IL-8 and RANTES production. NAC inhibited DEP-induced p38 MAP kinase activation and IL-8 and RANTES production. These results indicate that p38 MAP kinase plays an important role in the DEP-activated signaling pathway that regulates IL-8 and RANTES production by BECs and that the cellular redox state is critical for DEP-induced p38 MAP kinase activation leading to IL-8 and RANTES production.