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American Journal of Respiratory and Critical Care Medicine Vol 167. pp. 856-861, (2003)
© 2003 American Thoracic Society

Original Article

Apoptosis Signal-Regulating Kinase 1–Mediated Signaling Pathway Regulates Nitric Oxide–Induced Activator Protein-1 Activation in Human Bronchial Epithelial Cells

Itsuro Jibiki, Shu Hashimoto, Shuichiro Maruoka, Yasuhiro Gon, Atsushi Matsuzawa, Hideki Nishitoh, Hidenori Ichijo and Takashi Horie

First Department of Internal Medicine, and Section of Allergology and Immunology, High-Tech Research Center, Nihon University School of Medicine; and Laboratory of Cell Signaling, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan

Correspondence and requests for reprints should be addressed to Dr. Shu Hashimoto, First Department of Internal Medicine, Nihon University School of Medicine, 30-1 Oyaguchikamimachi, Itabashi-ku, Tokyo 173-8610, Japan. E-mail: shuh{at}med.nihon-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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.

Key Words: mitogen-activated protein kinase • airway inflammation • bronchial epithelium • activator protein-1 • nitric oxide

Nitric oxide (NO) is a molecular gas that is produced from the amino acid L-arginine by NO synthase (NOS) (1). NO displays a variety of biological functions including smooth muscle relaxation, neurotransmission, immune regulation, cellular differentiation, and host defense (1, 2). NO also may have antiinflammatory and proinflammatory effects (3–8). In bronchial asthma, the levels of exhaled NO and the expression of inducible NOS (iNOS) in the bronchial epithelial cells (BEC) are increased in patients with bronchial asthma compared with normal healthy subjects (9–11). However, the pathophysiologic roles of excessive produced endogenous NO in individuals with asthma are still unclear.

Airway epithelial cells that are exposed to various extracellular stimuli have the capacity to produce a variety type of biologically active molecules (12) and express various transcription factors, including nuclear factor-{kappa}B (NF-{kappa}B) and activator protein-1 (AP-1) (13–18). Both transcription factors play a role in the production of airway inflammation (13–18). NO has been shown to be capable of inducing NF-{kappa}B activation in airway epithelial cells (6); however, little is known about the effect of NO on AP-1 activation and the signaling pathway leading to NO-mediated AP-1 activation in BEC.

Many extracellular stimuli elicit specific biological responses through activation of mitogen-activated protein kinase (MAPK) cascades (19). Several subgroups of mammalian MAPK superfamily have been molecularly characterized: extracellular signal-regulated kinase (Erk), p38 MAPK, and c-Jun-NH2-terminal kinase (JNK). In the MAPK signaling cascades, MAPK kinase kinase (MAPKKK) activates MAPK kinase (MAPKK), which subsequently activates MAPK. Each MAPK is activated by distinct upstream kinases (19). Apoptosis signal-regulating kinase1 (ASK1) was identified as a member of the MAPKKK family that activates two different MAPK cascades, SEK1/MKK7-JNK and MKK3/MKK6-p38 MAPK pathways (20, 21). Overexpression of wild-type or the constitutively active form of ASK1 has been reported to induce apoptosis in various cell types (20–22), and the kinase-inactive mutant of ASK1 inhibited apoptosis induced by tumor necrosis factor-{alpha}, Fas ligation, anticancer drugs, or withdrawal of neurotrophic factors (20, 22–25). By contrast to the role of ASK1 in apoptosis, recent evidence has showed that ASK1 also functions the cell signaling molecule for regulating cell survival and differentiation (26, 27). ASK1 thus has a broad range of biological activities.

MAPK cascades are connected with the activation of various transcription factors (14, 28–31). AP-1 is known to be activated by MAPK, JNK, and p38 MAPK (29–31); however, little is known about a role of their upstream kinase, ASK1 in AP-1 activation in airway epithelial cells. In the present study, we first examined whether NO could induce AP-1 activation and analyzed the molecular mechanism in NO-induced AP-1 activation by focusing the role of ASK1 cascade.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and Reagents
Bronchial epithelial cell lines NCI-H292 and porcine aortic endothelial (PAE) cells (American Type Culture Collection, Rockville, MD) were grown in RPMI 1640 (Nissui Co. Ltd., Tokyo, Japan) supplemented with 10% heat-inactivated fetal calf serum (Mitsubishikasei Co. Ltd., Tokyo, Japan), streptomycin, and penicillin (Meiji Pharmaceutical Co. Ltd., Tokyo, Japan), and Ham's F12 medium (Nissui Co. Ltd.) supplemented with 10% heat-inactivated fetal calf serum, streptomycin, and penicillin, respectively. The reactive nitrogen generating species NOR-1 [(±)-(E)-Methyl-2-[(E)-hydroxykmino]-5-nitro-6-methoxy-3-hexeneamide] (32) and an NO scavenger, carboxyl-PTIO (2-(4-Caeboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxy-Oxide) (33) were obtained from Wako Pure Chemical Ltd. (Tokyo, Japan). NOR-1 and carboxy-PTIO were dissolved in dimethyl sulfoxide and PBS, respectively.

Cell Cultures
The cells were placed onto tissue culture plate (Falcon 1007; Falcon, Oxnard, CA) and cultured using culture medium at 37°C in humidified 5% CO2 atmosphere. When the cells were grown in subconfluent conditions, the culture medium was replaced with serum-free medium and the cells were cultured for 16 hours. To examine the effect of an NO scavenger on AP-1 activity, the cells that had been incubated with carboxy-PTIO for 1 hour were stimulated with NOR-1.

Transfection and Luciferase Reporter Assay
Serum-starved BEC were transiently transfected with the pcDNA3–ASK1-dominant negative expression vectors using FuGENE6 (Roche Diagnostics Corp., Indianapolis, IN). The total amount of cDNA was kept constant by supplementation with empty vector, pcDNA3 (Invitrogen, Carlsbad, CA). To generate dominant negative form of ASK1-stable transfected PAE cells, PAE cells were transfected with 2 µg each of plasmids pcDNA3-HA-ASK1KM or pcDNA3, using FuGENE6 (Roche) according to the manufacturer's instructions. After 16 hours, selection was initiated by adding 400 µg/ml G418 (Gibco BRL, Rockville, MD) to the culture medium. Independent colonies were cloned, and after screening by Western blotting using anti-HA antibody (3F10; Roche), positive clones were chosen and further analyzed. Every transfection included 500 ng of pAP-1-Luc reporter plasmid, together with either 5 ng of pRL-SV40-Renilla for normalization of transfection efficiency. After incubation for 24 hours, cells were stimulated with NOR-1. Then the cells were lysed in a luciferase lysis buffer (Promega, Madison, WI), and luciferase activity was determined using an assay kit (Promega) with TD-20/20 Luminometer (Promega). For controlling the efficiency of the transfection, the Renilla luciferase gene expression was monitored using pRL-SV40 and a dual luciferase system (Promega). Assays were performed in triplicate. The relative fold activation of luciferase was calculated.

Measurement of NO Concentrations
Amounts of NO in the culture supernatants were measured using total nitric oxide detection assay kit (Bio Visoin Research Products, Mountain View, CA).

Western Blot Analysis of JNK and p38 MAPK
ASK1 phoshorylation was analyzed using rabbit polyclonal antibody (Ab) to phosphorylated ASK1 (Phospho-ASK1) directed against a phosphorylated peptide fragment of mouse ASK1. Characterization of rabbit polyclonal Ab to ASK1 has been described previously (34, 35). JNK and p38 MAPK phosphorylation was analyzed according to manufacturer's instructions (Cell Signaling TECHNOLOGY, Beverly, MA) using an antiphosphorylated JNK Ab and an antiphosphorylated p38 MAPK Ab as described previously (36).

Statistical Analysis
Statistical significance was analyzed by using ANOVA. p Value less than 0.05 was considered significant. When statistical significance was reached, post hoc tests (Fischer's protected least significant difference, Scheff's F) were performed.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
NOR-1 Induces AP-1 Activity in Human Bronchial Epithelial Cells
First, we examined a concentration-dependent effect of NOR-1 on AP-1 activation in BEC. To this end, the cells that had been transiently transfected by pAP-1-Luc reporter plasmid were stimulated with various concentrations of NOR-1, and then AP-1–dependent luciferase gene activity was determined at 12 hours after stimulation with NOR-1 (Figure 1A) . AP-1 reporter activity in NOR-1–stimulated cells increased in a concentration-dependent manner. Next, we examined the kinetics of the effects of NOR-1 on AP-1 reporter activity. To this end, the cells were stimulated with 1 mM of NOR-1, and AP-1–dependent luciferase gene activity was determined at various times as indicated after stimulation with NOR-1 (Figure 1B). AP-1 reporter activity in NOR-1–stimulated cells increased in a time-dependent manner. To confirm the increases in AP-1 reporter activity resulted from NO release by an NO donor, NOR-1, the cells were preincubated with carboxy-PTIO, a NO scavenger, and then stimulated with NOR-1. Caroxy-PTIO attenuated NOR-1–mediated AP-1 activation (Figure 2) . Other NO generating agents, including SIN-1 and SNAP, also induced AP-1 activity. Fold increase in AP-1 reporter activity at 25 hours was 6.4 ± 0.4 (mean ± SD in three experiments) in SIN-1–stimulated BEC and 4.2 ± 0.4 (mean ± SD in three experiments) in SNAP-stimulated BEC. These results indicate that all three NO-generating agents, including NOR-1, SIN-1, and SNAP, activate AP-1.



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  		Figure 1. NOR-1 induces activator protein (AP)-1 activity in human bronchial epithelial cells (BEC). (A) BEC were stimulated with various concentrations with NOR-1, and AP-1 reporter activity was determined at 12 hours after stimulation. (B) BEC were stimulated with 1mM NOR-1, and AP-1 reporter activity was determined at the desired times as indicated. The results are expressed as means ± SD of six different experiments. *p < 0.01 compared with the NOR-1–unstimulated cells.

 


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  		Figure 2. Carboxy-PTIO, an NO scavenger, attenuates NOR-1–mediated AP-1 activation. BEC that had been preincubated with various concentrations of carboxy-PTIO for 1 hour were stimulated with 1 mM NOR-1, and then AP-1 reporter activity was determined at 12 hours after the stimulation with NOR-1. The results are expressed as means ± SD of six different experiments. *p < 0.01 compared with the NOR-1–unstimulated cells.

 
NOR-1 Induces ASK1, JNK, and p38 MAPK Phosphorylation
ASK1 regulates the activation of SEK1/MKK7-JNK and MKK3/MKK6-p38 MAPK pathways (19, 20). These pathways are connected with the activation of AP-1 (29–31). We next examined the role of ASK1-JNK/p38 MAPK cascades in NOR-1–induced AP-1 activation in BEC. First, we examined the threonine and tyrosine phosphorylation of ASK1, JNK, and p38 MAPK in NOR-1–stimulated BEC. BEC were stimulated with NOR-1, and ASK1, JNK, and p38 MAPK phosphorylation in the cells were immunoblotted at the desired times as indicated. Amounts of phosphorylated threonine and tyrosine of ASK1 increased at 10 minutes, were maximal at 30 minutes, and were thereafter sustained (Figure 3A , upper panel). Amounts of phosphorylated threonine and tyrosine of JNK increased at 10 minutes, were maximal at 30 minutes, and were thereafter sustained (Figure 3B, upper panel). Amounts of phosphorylated threonine and tyrosine of p38 MAPK increased at 10 minutes, were maximal at 15 minutes, and thereafter returned to basal levels (Figure 3C, upper panel). Lower panels of Figures 3A, 3B, and 3C showed that equal amounts of ASK1 protein, JNK protein, and p38 MAPK protein were immunoblotted with phosphorylation-independent ASK1-specific Ab, phosphorylation-independent JNK-specific Ab, and phosphorylation-independent p38 MAPK–specific Ab regardless of time of culture periods, respectively, indicating that NOR-1–induced JNK and p38 MAPK phosphorylation occurred in the absence of changes in JNK and p38 MAPK protein levels. Carboxy-PTIO attenuated NOR-1–mediated the phosphorylation of JNK and p38 MAPK (data not shown).



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  		Figure 3. NOR-1 induces the threonine and tyrosine phosphorylation of ASK1, JNK, and p38 MAPK in human BEC. BEC were stimulated with 1mM NOR-1 for the desired times as indicated. The cell lysate containing 10 µg of protein separated by 15% SDS-PAGE was electophoretically transferred to nitrocellulose membrane and the membrane was blotted with an antiphosphorylated threonine and tyrosine of ASK1 Ab (p-ASK1; upper panel of A), an antiphosphorylated threonine and tyrosine of JNK Ab (p-JNK; upper panel of B) or an antiphosphorylated threonine and tyrosine of p38 MAPK Ab (p-p38 MAPK; upper panel of C). Then it was incubated with the HRP-conjugated anti-rabbit IgG Ab and HRP-conjugated anti-biotin Ab to detect biotinylated protein markers. Blots were incubated with ECL solution for 1 minute and exposed on KODAK XAR film. Blots were stripped and reprobed using phosphorylation-state independent ASK1 Ab (ASK1; lower panel of A), phosphorylation-state independent JNK Ab (JNK; lower panel of B) or phosphorylation-state independent p38 MAPK Ab (p38 MAPK; lower panel of C). The amounts of phosphorylated ASK1, JNK, and p38 MAPK were quantified by NIH image analyzer and are presented as the amounts of phosphorylated ASK1, JNK, and p38 MAP kinase proteins relative to control cells treated without NOR-1 (1.0). The fold increases in amounts of phosphorylated ASK1, JNK, and p38 MAPK proteins are indicated below. Three identical experiments independently performed gave similar results.

 
ASK1 Regulates NOR-1–induced AP-1 Activation
In the next, to examine a role of ASK1 in NOR-1–mdediated AP-1 activation, the BEC were transiently transfected by the dominant negative form of ASK1. Transient transfection of a dominant negative form of ASK1 attenuated NOR-1–mediated AP-1 activation in BEC (Figure 4) . To further characterize the role of ASK1 in NOR-1–induced AP-1 activation, we used the dominant negative form of ASK1-stable transfected PAE. The dominant negative form of ASK1-transfected PAE and the parental PAE cells were stimulated with NOR-1. JNK and p38 MAPK phosphorylation was analyzed at the time as indicated, and AP-1 activity was determined at 24 hours after NOR-1 stimulation. Amounts of phosphorylated JNK and p38 MAPK were lower in the dominant negative form of ASK1-stable transfected PAE than those in the parental PAE (Figure 5) , showing that JNK and p38 MAPK phosphorylation was depressed in the dominant negative form of ASK1-stable transfected PAE. AP-1 reporter activity is lower in the dominant negative form of ASK1 than those in the parental PAE (Figure 6) , showing that AP-1 activity was depressed in the dominant negative form of ASK1-stable transfected PAE. These results indicate that ASK-1 is an upstream kinase of JNK and p38 MAPK activation and ASK-1-JNK/p38 MAPK cascade regulates NOR-1–induced AP-1 activation. Amounts of NO in NOR-1–unstimulated and –stimulated dominant negative form of ASK1-transiently transfected BEC at 10 minutes after stimulation were 0.8 ± 0.1 M and 1.3 ± 0.1 M, respectively. Amounts of NO in NOR-1–unstimulated and –stimulated empty pcDNA3 vector-transiently transfected BEC at 10 minutes after stimulation were 0.8 ± 0.1 M and 1.3 ± 0.1 M, respectively. Similarly, there was no difference in amounts of generated NO between the dominant negative form of ASK1-stable transfected PAE and the parental PAE (data not shown). These results indicated that NO was similarly generated regardless of a dominant negative form of ASK1 in the cells. The total number of the cells, cell viability determined by trypan blue exclusion dye and total cell protein at the end of the culture period of each experiment indicated in Figures 1 through 6 did not differ with culture conditions (data not shown).



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  		Figure 4. AP-1 activity is depressed in the dominant negative form of ASK1-transfected human bronchial epithelial cells. BEC were transiently co-transfected with the pcDNA3–ASK1-dominant negative expression vectors or empty pcDNA3 vector and pAP-1-Luc reporter plasmid. After 24 hours with the transfection, BEC were stimulated with 1mM NOR1 and AP-1 activity was determined at 12 hours after the stimulation with NOR-1. The results are expressed as means ± SD of six different experiments. *p < 0.05 compared with the NOR-1–stimulated dominant negative form of ASK1-untransfected cells. **p < 0.01 compared with the NOR-1–stimulated dominant negative form of ASK1-untransfected cells.

 


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  		Figure 5. JNK and p38 MAPK phosphorylation is depressed in the dominant negative form of ASK1-stable transfected PAE. The parental PAE (A and C) and the dominant negative form of ASK1-stable transfected PAE (B and D) were stimulated with 1 mM NOR-1 for the desired times as indicated. The cell lysate containing 10 µg of protein separated by 15% SDS-PAGE was electophoretically transferred to nitrocellulose membrane, and the membrane was blotted with an antiphosphorylated threonine and tyrosine of JNK Ab (phospho-JNK; upper panels of A and B) or an antiphosphorylated threonine and tyrosine of p38 MAPK Ab (phospho-p38 MAPK; upper panels of C and D). Next, it was incubated with the HRP-conjugated anti-rabbit IgG Ab and HRP-conjugated anti-biotin Ab to detect biotinylated protein markers. Blots were incubated with ECL solution for 1 min and exposed on KODAK XAR film. Blots were stripped and reprobed using phosphorylation-state independent JNK Ab (JNK; lower panels of A and C) or phosphorylation-state independent p38 MAPK Ab (p38 MAPK; lower panels of B and D). The amounts of phosphorylated JNK and p38 MAPK were quantified by NIH image analyzer and are presented as the amounts of phosphorylated JNK and p38 MAP kinase proteins relative to control cells treated without NOR-1 (1.0). The fold increases in amounts of phosphorylated JNK and p38 MAPK proteins are indicated below. Three identical experiments independently performed gave similar results.

 


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  		Figure 6. AP-1 activity is depressed in the dominant negative form of ASK1-transfected PAE. The parental PAE and the dominant negative form of ASK1-stable transfected PAE were stimulated with 1 mM NOR-1 were transiently transfected with pAP-1-Luc reporter plasmid or empty pcDNA3 vector. After 24 hours with the transient transfection, the parental PAE and the dominant negative form of ASK1-stable transfected PAE were stimulated with 1 mM NOR-1, and AP-1 activity was determined at 12 hours with the stimulation with NOR-1. The results are expressed as means ± SD of six different experiments. *p < 0.01 compared with the NOR-1–stimulated parental PAE cells.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we examined the effect of NO in inducing AP-1 activation in human bronchial epithelial cells and analyzed the intracellular signal regulating NO-mediated AP-1 activation determined by AP-1–dependent luciferase gene activity. The results showed that (1) NOR-1 induced AP-1 reporter activity, and carboxy-PTIO attenuated NOR-1–mediated AP-1 activation; (2) NOR-1 induced increases in ASK1, JNK, and p38 MAPK phosphorylation; and (3) transient transfection of the dominant negative ASK1 attenuated NOR-1–mediated AP-1 activation in BEC. To further characterize the role of ASK-1-JNK/p38 MAPK cascade, the dominant negative forms of ASK1-stable transfected PAE were used. JNK and p38 MAPK phosphorylation, and AP-1 activity, were depressed in the dominant negative form of ASK1-stable transfected PAE. The transfection of the dominant negative form of ASK1 did not completely depress JNK and p38 MAPK phosphorylation and AP-1 activity. These results indicate that NO is capable of inducing AP-1 activation, and that ASK1-JNK/p38 MAPK cascade, at least in part, regulates AP-1 activation in NO-stimulated BEC.

AP-1 consists of homodimers and heterodimers of Jun (c-Jun, Jun, JunB, JunD), Fos (c-Fos, FosB, Fra-1, Fra-2), or the activating transcription factor (ATF-2, ATF-3) proteins which bind to a common DNA site, the AP-1 site. AP-1 activity in response to extracellular stimuli is regulated at two major levels. At a first level, the transcription of jun and fos genes are upregulated by extracellular stimuli. At a second level, the activity of preexisting AP-1 factors is posttranscriptionally modified by their phosphorylation (28). In addition to the role of JNK in AP-1 activation, recent evidence has shown that p38 MAPK plays a role in AP-1 activation (19, 30, 31). MAP kinase cascades are connected with the activation of AP-1. c-Jun, JunD, and ATF-2 are phosphorylated by JNK, resulting in an increased transactivation potential of the factors (28). In the present study, we analyzed the role of ASK1-JNK/p38 MAPK cascade in NOR-1–induced AP-1 activation in BEC. ASK1 is an upstream kinase kinase of JNK and p38 MAPK (18, 19). JNK and p38 MAPK are activated through ASK1 in response to various extracellular stimuli, including hydrogen peroxide, tumor necrosis factor-{alpha}, and microtubule-disrupting agents (20, 21, 24). We examined the role of ASK1-JNK and p38 MAPK cascade in NOR-1–induced AP-1 activation by transient transfection of a dominant negative form of ASK1 in BEC. The results showed that transient transfection of a dominant negative form of ASK1 attenuated NOR-1–mediated AP-1 activation in BEC. To further characterize the role of ASK1-JNK and p38 MAPK cascade in NOR-1–induced AP-1 activation, we use the dominant negative form of ASK1-stable transfected PAE. JNK and p38 MAPK phosphorylation and AP-1 activation were depressed in the dominant negative form of ASK1-stable transfected PAE. These results indicate that JNK and p38 MAPK is activated through ASK1 in response to NOR-1, and NOR-1–activated ASK1-JNK/p38 MAPK cascade regulates AP-1 activation.

Multiple inflammatory cells, lung structural cells including BEC and cytokines and mediators participate in the production of airway inflammation seen in bronchial asthma. The role of transcription factor, AP-1, and NF-{kappa}B in the production of airway inflammation has been documented (13–18). An increased expression and activity of AP-1 and NF-{kappa}B is seen in BEC from individuals with bronchial asthma compared with normal healthy subjects (13–18). Activity and expression of these transcription factors are upregulated by proinflammatory stimuli, including NO (8, 15–18). Corticosteroid, including inhaled corticosteroid, reduces the activity and the expression of AP-1 and NF-{kappa}B, but the activity of AP-1 and NF-{kappa}B in individuals with corticosteroid-resistant asthma cannot be suppressed by corticosteroid (14, 15, 37). These transcription factors participate to various extents in the inducible expression of the gene encoding various cytokines including RANTES and eotaxin involved in the production of allergic inflammation. Therefore, it is important to reduce the activity and the expression of AP-1 and NF-{kappa}B. Although the precise role of NO in AP-1 activation in vivo remain to be clarified, NOS inhibitor might be useful for the treatment of bronchial asthma, especially in individuals with corticosteroid-resistant asthma. In addition, therapeutic intervention in inhibiting JNK and p38 MAP kinase activation may be beneficial in reducing AP-1 activity in BEC.

From the data presented here, we conclude that ASK1-JNK/p38 MAPK-dependent pathway regulates the reactive nitrogen generating species, NOR-1–induced AP-1 activation. Our results with the role of ASK1-JNK/p38 MAPK-dependent pathway in NO-induced AP-1 activation is important in understanding the molecular mechanism in AP-1 activation seen in airway epithelial cells of individuals with bronchial asthma, and a strategy of attenuating airway inflammation by the specific inhibitor of ASK1 cascade may produce beneficial effects in controlling airway inflammation.


   FOOTNOTES
 
Supported in part by Grant-in-Aid for High-Tech Research Center from the Japanese Ministry of Education, Science, Sports, and Culture to Nihon University and for General Scientific Research from the Ministry of Education of Japan.

Received in original form April 29, 2002; accepted in final form December 2, 2002


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