Involvement of p42/p44 MAPK, p38 MAPK, JNK, and NF-
B in IL-1
-induced VCAM-1 expression in human tracheal smooth muscle cells
Chien-Chun Wang,1
Wei-Ning Lin,2
Chiang-Wen Lee,2
Chih-Chung Lin,3
Shue-Fen Luo,4
Jong-Shyan Wang,5 and
Chuen-Mao Yang1,2
1Graduate Institute of Natural Products, Departments of 2Pharmacology, 3Anesthetics, and 4Internal Medicine, and 5Graduate Institute of Rehabilitation Science, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan
Submitted 11 June 2004
; accepted in final form 20 September 2004
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ABSTRACT
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Interleukin-1
(IL-1
) has been shown to induce the expression of adhesion molecules on airway epithelial and smooth cells and contributes to inflammatory responses. Here, the roles of mitogen-activated protein kinases (MAPKs) and nuclear factor-
B (NF-
B) pathways for IL-1
-induced vascular cell adhesion molecule (VCAM)-1 expression were investigated in human tracheal smooth muscle cells (HTSMC). IL-1
induced expression of VCAM-1 protein and mRNA in a time-dependent manner, which was significantly inhibited by inhibitors of MEK1/2 (U0126 and PD-98059), p38 (SB-202190), and c-Jun NH2-terminal kinase (JNK; SP-600125). Consistently, IL-1
-stimulated phosphorylation of p42/p44 MAPK, p38, and JNK was attenuated by pretreatment with U0126, SB-202190, or SP-600125, respectively. IL-1
-induced VCAM-1 expression was significantly blocked by the specific NF-
B inhibitors helenalin and pyrrolidine dithiocarbamate. As expected, IL-1
-stimulated translocation of NF-
B into the nucleus and degradation of I
B-
were blocked by helenalin but not by U0126, SB-202190, or SP-600125. Moreover, the resultant enhancement of VCAM-1 expression increased the adhesion of polymorphonuclear cells to a monolayer of HTSMC, which was blocked by pretreatment with helenalin, U0126, SB-202190, or SP-600125 before IL-1
exposure or by anti-VCAM-1 antibody. Together, these results suggest that in HTSMC, activation of p42/p44 MAPK, p38, JNK, and NF-
B pathways is essential for IL-1
-induced VCAM-1 gene expression. These results provide new insight into the mechanisms of IL-1
action that cytokines may promote inflammatory responses in airway disease.
interleukin-1
; mitogen-activated protein kinase; c-Jun NH2-terminal kinase; nuclear factor-
B; vascular cell adhesion molecule-1
THE RECRUITMENT AND ADHESION of circulating polymorphonuclear cells (PMN) to the vascular endothelium play a critical role in the inflammation response. This event is mediated through the expression of adhesive molecules on the cell surface of endothelial cells and PMN (30). The adhesion molecules included intercellular adhesion molecule-1 (ICAM-1), a ligand for the leukocyte
2-integrins, which mediates the tight adhesive binding of PMN and thus facilitates PMN migration across the vascular endothelial barrier (46). Adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1), are also inducible cell surface glycoproteins of several cell types and play an important role in a number of inflammatory and immune responses. Upregulation of ICAM-1 expression on cytokine-triggered vascular endothelial cells enhances the targeted transmigration of PMN into extravascular space of inflammation (16, 46). In airways, to reach the submucosa and airway lumen, circulating PMN must first be recruited across the vascular endothelium (11, 37) and then migrate through the interstitial matrix before interacting with airway epithelium. Thus similar processes that govern PMN adhesion to lung airway epithelial cells may occur and contribute to the damage to these cells seen in inflammatory responses of asthma (8, 36, 49). This event is crucial in the development of allergic inflammation and is mediated by adhesion molecules and cytokines (19, 27, 36). During these interactions, PMN and lung tissue undergo cytokine-specific upregulation of adhesion molecules (22).
Although the roles of cytokines and adhesion molecules in PMN adhesion to endothelial cells are well described, little is known about the interaction between PMN and tracheal smooth muscle cells (TSMC). Cytokines are potent immunoregulatory and proinflammatory mediators secreted by a variety of cell responses to infection, activated by lymphocyte products, microbial toxins, and other stimuli (3, 40). Elevated levels of proinflammatory cytokines, including IL-1
in the bronchoalveolar lavage fluid, have been detected in allergic asthmatic patients (10, 34). IL-1
exerts as a potent stimulus in inflammatory responses through upregulation of many gene expressions, including cytokines, chemokines, proteases, cyclooxygenase (COX), and adhesion molecules. The extent and duration of these gene expressions are correlated with the severity of inflammatory processes (14). The expression of VCAM-1 induced by IL-1
may be integrated to the signaling networks that augment airway inflammation by recruiting and activating leukocytes. Several reports have described the induction of VCAM-1 by inflammatory cytokines such as IL-1
, TNF-
, and IFN-
on human umbilical vein endothelial cells, pulmonary artery endothelial cells (38), intestinal epithelial cells (26), keratocytes (16), and renal tubular epithelial cells (23). Moreover, the expression of ICAM-1 and other genes appears to be highly regulated by a number of mitogen-activated protein kinases (MAPKs) and NF-
B in a variety of cell types (1, 2, 4, 7, 12, 13).
There are at least three distinct and parallel MAPK pathways that have been characterized, which include p42/p44 MAPK (9), p38 MAPK (21), and c-Jun NH2-terminal kinase (JNK) (28), activated by phosphorylation of a tyrosine and a threonine residue catalyzed by a dual specificity MAPK kinase. Activation of MAPKs exerts distinct cellular responses mediated by phosphorylation of specific target proteins (33). Although cytokines such as IL-1
are reported to activate all of these MAPKs (20, 47), the relationship between the activation of these pathways and expression of adhesion molecules or other genes has been controversial. IL-1
has been shown to induce ICAM-1 expression mediated through activation of NF-
B, but not p42/p44 MAPK, p38, and JNK in A549 cells (12). In human retinal pigment epithelial cells, activation of p42/p44 MAPK, p38, and NF-
B is required for IL-1
-induced chemokine expression (7). The discrepancies in these previous reports imply that there are divergent pathways leading to ICAM-1 expression induced by IL-1
, depending on the nature of stimulus, cell types, and target genes. In addition, our previous studies have shown that activation of MAPKs is involved in cell proliferation and induction of bradykinin receptor and COX-2 expression by IL-1
in TSMCs (5355). Therefore, whether activation of these MAPK pathways by IL-1
is also linked to VCAM-1 expression needs to be determined in human tracheal smooth muscle cells (HTSMC). In addition, it is of interest that many of the genes regulated by MAPKs are dependent on NF-
B for transcription (7, 12, 39). NF-
B has also been shown to involve in ICAM-1 gene expression at the transcriptional level in various cell types (12, 44).
In addressing these questions, the experiments were performed to investigate the roles of p42/p44 MAPK, p38, JNK, and NF-
B in IL-1
-induced VCAM-1 mRNA and protein production in HTSMC. We found that coactivation of p42/p44 MAPK, p38, and JNK is required for maximal induction of VCAM-1 gene expression by HTSMC. These findings suggest that the increased expression of VCAM-1 correlates with increased adhesion of PMN to IL-1
-challenged HTSMC, at least in part, mediated through these MAPKs and NF-
B signaling pathways. These results provide new insight into the mechanisms of IL-1
action, supporting the hypothesis that cytokines may contribute to leukocyte/HTSMC interaction and promote inflammatory responses involved in the development of airway disease.
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MATERIALS AND METHODS
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Materials.
DMEM/F-12 medium, FBS, and TRIzol were purchased from Invitrogen (Carlsbad, CA). Hybond C membrane, enhanced chemiluminescence (ECL) Western blotting detection system, and Hyperfilms were from Amersham Biosciences (Buckinghamshire, UK). The polyclonal antibodies VCAM-1, I
B-
, and NF-
B p65 were from Santa Cruz (Santa Cruz, CA). Anti-GAPDH antibody was from Biogenesis (Boumemouth, UK). PhosphoPlus p42/p44 MAPK, p38 MAPK, and stress-activated protein kinase/JNK antibody kits were from New England Biolabs (Beverly, MA). U0126, PD-98059, SB-202190, SP-600125, pyrrolidine dithiocarbamate (PDTC), and helenalin were from Biomol (Plymouth Meeting, PA). Bicinchoninic acid (BCA) protein assay kit was from Pierce (Rockford, IL). Enzymes and other chemicals were from Sigma (St. Louis, MO).
Cell culture.
HTSMC were isolated from human trachea obtained from lung transplant donors who were healthy and suffered from brain trauma in the 24 h during organ transplantation. All the protocols followed the guidance of the Institutional Human and Animal Care and Use Committee of Chang Gung University. Tracheal smooth muscle strips denuded of mucosa and connective tissue were cut into small pieces and placed in six-well culture plates. These explants were grown in DMEM/F-12 containing 10% (vol/vol) FBS and antibiotics (100 U/ml penicillin G, 100 µg/ml streptomycin, and 250 ng/ml fungizone) at 37°C in a humidified 5% CO2 atmosphere as described in a previous study (29). When the cultures reach confluence (14 days), cells were treated with 0.05% (wt/vol) trypsin/0.53 mM EDTA for 5 min at 37°C. The cell suspension was diluted with DMEM/F-12 containing 10% FBS to a concentration of 2 x 105 cells/ml. The cell suspension was plated onto (1 ml/well) 12-well culture plates and (10 ml/dish) 10-cm culture dishes for the measurement of protein expression and mRNA accumulation, respectively. Culture medium was changed after 24 h and then every 3 days.
To characterize the isolated and cultured HTSMC and to exclude contamination by epithelial cells and fibroblasts, the cells were identified by an indirect immunofluorescent staining method using a monoclonal antibody of
-actin. We found that >95% of the culture cells reacted positively with antibody generated against
-actin (data not shown). Experiments were performed with cells from passages 38.
Preparation of cell extracts and Western blot analysis.
HTSMC were plated onto 12-well culture plates and made quiescent at confluence by incubation in serum-free DMEM/F-12 for 24 h. Growth-arrested cells were incubated with or without different concentrations of IL-1
at 37°C for the indicated times. When inhibitors were used, they were added 1 h before the application of IL-1
. After being incubated, the cells were then rapidly washed with ice-cold PBS, scraped, and collected by centrifugation at 1,000 g for 10 min. The collected cells were lysed with ice-cold lysis buffer containing 25 mM Tris·HCl, pH 7.4, 25 mM NaCl, 25 mM NaF, 25 mM sodium pyrophosphate, 1 mM sodium vanadate, 2.5 mM EDTA, 2.5 mM EGTA, 0.05% (wt/vol) Triton X-100, 0.5% (wt/vol) SDS, 0.5% (wt/vol) deoxycholate, 0.5% (wt/vol) Nonidet P-40, 5 µg/ml leupeptin, 5 µg/ml aprotinin, and 1 mM PMSF. The lysates were centrifuged at 45,000 g for 1 h at 4°C to yield the whole cell extract. The protein concentration was determined by the BCA reagents according to the manufacturers instructions. Samples from these supernatant fractions (30 µg of protein) were denatured and subjected to SDS-PAGE using a 10% running gel. Proteins were transferred to nitrocellulose membrane, and the membrane was incubated successively at room temperature with 5% (wt/vol) BSA in TTBS [50 mM Tris·HCl, 150 mM NaCl, and 0.05% (wt/vol) Tween 20, pH 7.4] for 1 h. Membranes were incubated overnight at 4°C with the anti-VCAM-1 or anti-GAPDH antibody used at a dilution of 1:2,000 in TTBS. Membranes were washed with TTBS four times for 5 min each and incubated with a 1:1,500 dilution of anti-goat or anti-mouse horseradish peroxidase antibody for 1 h. After each incubation, the membrane was washed extensively with TTBS. The immunoreactive bands detected by ECL reagents were developed by Hyperfilm-ECL.
Total RNA extraction and RT-PCR analysis.
Total RNA was isolated from HTSMC treated with IL-1
for the indicated times in 10-cm culture dishes with TRIzol according to the protocol of the manufacturer. RNA concentration was spectrophometrically determined at 260 nm. First-strand cDNA synthesis was performed with 2 µg of total RNA using random hexamers as primers in a final volume of 20 µl (5 µg/µl random hexamers, 1 mM dNTPs, 2 U/µl RNasin, and 10 U/µl Moloney murine leukemia virus reverse transcriptase). The reaction was carried out at 37°C for 60 min. cDNAs encoding
-actin and VCAM-1 were amplified from 35 µl of the cDNA reaction mixture using specific gene primers. Oligonucleotide primers for
-actin and VCAM-1 were as follows:
-actin, 5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3' (sense), 5'-CTAGAAGCATTTGCGGTGGACGATG-3' (antisense); VCAM-1, 5'-GGAACCTTGCAGCTTACAGTGACAGAGCTCCC-3' (sense), 5'-CAAGTCTACATATCACCCAAG-3' (antisense). The amplification profile includes 1 cycle of initial denaturation at 94°C for 5 min, 30 cycles of denaturation at 94°C for 1 min, primer annealing at 62°C for 1 min, extension at 72°C for 1 min, and then 1 cycle of final extension at 72°C for 5 min. The expression of
-actin was to be used as an internal control for the assay of a constitutively expressed gene.
NF-
B translocation.
HTSMC were seeded in a 10-cm dish. After they reached 90% confluence, the cells were starved for 24 h in serum-free DMEM/F-12 medium. After stimulation with 1.5 ng/ml of IL-1
, the cells were washed one time with ice-cold PBS, 200 µl of homogenization buffer A (20 mM Tris·HCl, pH 8.0, 10 mM EGTA, 2 mM EDTA, 2 mM dithiothreitol, 1 mM PMSF, 25 µg/ml aprotinin, and 10 µg/ml leupeptin) was added to each dish, and the cells were scraped into a 1.5-ml tube with a rubber policeman. The suspension was sonicated for 10 s at output 4 with a sonicator (Ultrasonics) and centrifuged at 8,000 rpm for 5 min at 4°C. The supernatant was collected as the cytosol fraction and the pellet as the nuclear fraction. The pellet was resuspended in 300 µl of homogenization buffer B (1% Triton X-100 in buffer A) and sonicated for 10 s. The suspension was centrifuged at 15,000 rpm for 15 min at 4°C. The supernatant was collected as a nuclear lysate fraction. The protein concentration of each sample was determined by the BCA reagents. Samples from these supernatant fractions (30 µg of protein) were denatured and subjected to SDS-PAGE using a 10% (wt/vol) running gel. Proteins were transferred to nitrocellulose membrane, and the membrane was incubated successively at room temperature with 1% (wt/vol) BSA in TTBS for 1 h. The translocation of NF-
B was identified and quantified by Western blot analysis using I
B-
or NF-
B (p65) antibody. The immunoreactive bands detected by ECL reagents were developed by Hyperfilm-ECL.
Immunofluorescence staining.
HTSMC were plated on six-well culture plates with coverslips. Cells were further cultured in serum-free DMEM/F-12 for 24 h and treated with 1.5 ng/ml of IL-1
. After being washed two times with ice-cold PBS, the cells were fixed with 4% (wt/vol) paraformaldehyde in PBS for 30 min and then permeabilized with 0.3% Triton X-100 in PBS for 15 min. The staining was performed by incubating with 10% normal goat serum in PBS for 30 min followed by incubating with primary anti-NF-
B p65 polyclonal antibody (1:100 dilution) for 1 h in PBS with 1% BSA, washing three times with PBS, incubating for 1 h with FITC-conjugated goat anti-rabbit antibody (1:100 dilution) in PBS with 1% BSA, washing three times with PBS, and finally mounting with aqueous mounting medium. The images were observed under a fluorescence microscope (Nikon, Optiphoto 2/EFD2).
Neutrophil adhesion assay.
Peripheral blood PMN were isolated from whole venous blood by dextran sedimentation followed by density separation over Ficoll-Hypaque and hypotonic lysis. The PMN were then resuspended in Tyrode-HEPES buffer (128 mM NaCl, 2.7 mM KCl, 0.5 mM MgCl2, 0.36 mM NaH2PO4, 2 mM CaCl2, 12 mM NaHCO3, and 10 mM HEPES, pH 7.4) and adjusted to 1 x 107 cells/ml. PMN were used within 4 h of purification.
Leukocyte-HTSMC adhesion was measured by a parallel plate chamber according to the methods described previously (51). HTSMC grew on glass coverslips pretreated with 0.15 ng/ml of IL-1
for 24 h. After the chamber was assembled, it was placed on the stage of an inverted microscope (Nikon, Tokyo, Japan) equipped with a charge-coupled device video camera (Mintron Enterprise). The inlet of the chamber was then connected to a perfusion system (KD Scientific, New Hope, PA), and PMN were gently infused into the chamber and kept there for 30 min at 37°C. The flow chamber was flushed with Tyrode-HEPES buffer for 5 min at a flow rate of 0.4 ml/min, corresponding to surface shear stress of 2 dyn/cm2. The nonadherent PMN were washed away from the slide. After flow perfusion, the number of neutrophils adhering to HTSMC was analyzed with an inverted light microscope (Nikon) connected to an image analysis system (Moti Images plus 2.0, Micro-Optic Industrial Group).
Analysis of data.
Concentration-effect curves were fitted, and EC50 values were estimated using the GraphPad Prism Program (GraphPad, San Diego, CA). Data were expressed as means ± SE and analyzed with a one-way ANOVA to make comparisons with the Bonferroni test at a P < 0.05 level of significance.
 |
RESULTS
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IL-1
-induced de novo VCAM-1 protein and gene expression.
To determine the effect of IL-1
on the expression of VCAM-1 protein and mRNA, we treated HTSMC with various concentrations of IL-1
for the indicated times before harvesting them for immunoblot analysis or isolation of total RNA for RT-PCR. The amount of de novo synthesis of VCAM-1 protein was determined using Western blot analysis. As shown in Fig. 1A, IL-1
induced VCAM-1 protein expression in a time- and concentration-dependent manner. There was a significant increase in this response being observed within 4 h, when HTSMC were exposed to these concentrations of IL-1
. A maximal response was achieved within 6 h and then slightly declined at 24 h. The amount of VCAM-1 protein expression was increased with increasing concentrations of IL-1
(Fig. 1B). The blot was stripped and reprobed with an anti-GAPDH antibody to demonstrate equivalent amount of GAPDH expression.
To further examine whether the effect of IL-1
on VCAM-1 expression was involved at the level of transcription, VCAM-1 mRNA was determined by RT-PCR. As shown in Fig. 1C, IL-1
induced VCAM-1 mRNA accumulation in a time-dependent manner. A maximal response was obtained within 48 h during the period of observation.
IL-1
induces VCAM-1 expression via p42/p44 MAPK phosphorylation.
Next, we investigated whether IL-1
-induced VCAM-1 expression was mediated via p42/p44 MAPK in HTSMC. Data in Fig. 2A show that pretreatment with the MEK1/2 inhibitors U0126 (18) or PD-98059 (15) for 1 h before exposure to IL-1
for 24 h caused an attenuation of VCAM-1 expression in a concentration-dependent manner. A concentration of 1 µM U0126 or 10 µM PD-98059 almost completely inhibited VCAM-1 expression induced by IL-1
(P < 0.05, n = 3, compared with cells exposed to IL-1
alone).
To determine whether p42/p44 MAPK phosphorylation is necessary for the induction of VCAM-1 expression upon exposure to IL-1
, activation of these kinases was assayed using an antibody specific for the phosphorylated, active forms of p42/p44 MAPK determined by Western blotting. As shown in Fig. 2B, IL-1
stimulated a transient phosphorylation of p42/p44 MAPK in HTSMC. The maximal response was obtained within 5 min (P < 0.05, n = 3, compared with the basal level) and then declined to the basal level within 30 min. Activation of p42/p44 MAPK is mediated through an upstream component of MAPK kinases, MEK1/2. To further examine the involvement of MEK1/2 in p42/p44 MAPK phosphorylation, HTSMC were pretreated with U0126 and then stimulated with IL-1
for different time periods. Pretreatment with U0126 (0.1 µM) for 1 h sustained inhibition of IL-1
-stimulated p42/p44 MAPK phosphorylation (P < 0.05, n = 3, compared with cells exposed to IL-1
alone; Fig. 2B). Together, these results suggest a link between activation of the MEK1/2-p42/p44 MAPK pathway and induction of VCAM-1 expression by IL-1
in HTSMC.
IL-1
-induced VCAM-1 expression via p38 MAPK phosphorylation.
To determine whether p38 MAPK was involved in IL-1
-induced VCAM-1 expression in HTSMC, a p38 MAPK inhibitor (SB-202190, Ref. 24) was used. As shown in Fig. 3A, pretreatment with SB-202190 resulted in a significant attenuation of VCAM-1 expression (
64%; P < 0.05, n = 3, compared with cells exposed to IL-1
alone) revealed by Western blotting. To ensure IL-1
stimulated p38 MAPK phosphorylation, activation of this kinase was assayed using an antibody specific for the phosphorylated, active forms of p38 MAPK by Western blot. As shown in Fig. 3B, IL-1
stimulated a time-dependent phosphorylation of p38 MAPK with a maximal response within 10 min (P < 0.05, n = 3, compared with the basal level) that then slightly declined in HTSMC. IL-1
-stimulated p38 MAPK phosphorylation was partially inhibited (
51%, P < 0.05, n = 3, compared with cells exposed to IL-1
alone) by 10 µM SB-202190 (Fig. 3C). These results suggest that activation of p38 MAPK was partially involved in IL-1
-induced VCAM-1 expression in HTSMC.
IL-1
-induced VCAM-1 expression via JNK phosphorylation.
To determine whether JNK was also involved in IL-1
-induced VCAM-1 expression in HTSMC, the pharmacological inhibitor of JNK (SP-600125, Ref. 6) was used. As shown in Fig. 4A, pretreatment of HTSMC with SP-600125 (1 µM) effectively blocked VCAM-1 expression (
72%; P < 0.05, n = 3, compared with cells exposed to IL-1
alone) in a concentration-dependent manner. In addition, to ensure IL-1
stimulated JNK phosphorylation, activation of this kinase was assayed using an antibody specific for the phosphorylated, active forms of JNK by Western blot. As shown in Fig. 4B, IL-1
stimulated a time-dependent phosphorylation of JNK with a maximal response within 30 min (P < 0.05, n = 3, compared with the basal level) that then slightly declined in HTSMC. To further examine the effect of SP-600125 on JNK phosphorylation, HTSMC were pretreated with SP-600125 and then stimulated with IL-1
for different time periods. Pretreatment of HTSMC with 0.1 µM SP-600125 also resulted in a significant attenuation of JNK phosphorylation (P < 0.05, n = 3, compared with cells exposed to IL-1
alone). These results suggest that activation of JNK may be involved in VCAM-1 expression induced by IL-1
.
NF-
B inhibitors suppress IL-1
-induced VCAM-1 expression.
Inflammatory responses after stimulation with cytokines such as IL-1
are highly dependent on activation of the NF-
B transcription factor. Moreover, NF-
B is one of the major mediators of the intracellular functions of IL-1
. In addition, IL-1
has been shown to be involved in VCAM-1 gene expression through NF-
B cascade (12, 41). Therefore, the involvement of NF-
B activation in VCAM-1 expression following stimulation with IL-1
was further confirmed using pharmacological inhibitors in HTSMC. For this purpose, helenalin, a specific sesquiterpene lactone compound known to inhibit NF-
B (32) and PDTC, was used. As shown in Fig. 5A, pretreatment of HTSMC with either helenalin or PDTC for 1 h before exposure to IL-1
caused an attenuation of VCAM-1 protein expression (P < 0.05, n = 3, compared with cells exposed to IL-1
alone) in a concentration-dependent manner. At the highest concentrations used, both helenalin and PDTC almost completely inhibited VCAM-1 protein expression induced by IL-1
in HTSMC.

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Fig. 5. Effect of NF- B inhibitor on IL-1 -stimulated expression of VCAM-1 in HTSMC. Cells were preincubated with helenalin (HLN) for 1 h and then incubated with IL-1 . A: after a 24-h incubation, the cell lysates were subjected to 10% SDS-PAGE and transferred to nitrocellulose membrane to determine the level of VCAM-1 protein expression as described in Fig. 1. B: time dependence of IL-1 -induced NF- B translocation and I B- degradation. Cells were treated with 1.5 ng/ml of IL-1 for 30 and 60 min or in the presence of U0126 (1 µM), SB-202190 (30 µM), SP-600125 (10 µM), or helenalin (10 µM) for 1 h and then treated with 1.5 ng/ml of IL-1 for 30 min. Cells were harvested and centrifuged to prepare cytosolic and nuclear fractions. The resultant fractions were subjected to 10% SDS-PAGE and analyzed with anti-NF- B (p65), anti-I B- , or GAPDH (as a control) antibody as described in MATERIALS AND METHODS. C: nuclear translocation of NF- B determined by immunofluorescence staining. HTSMC were pretreated with or without U0126 (1 µM), SB-202190 (30 µM), SP-600125 (10 µM), or helenalin (10 µM) for 1 h and were then stimulated with 1.5 ng/ml of IL-1 for 30 min. Cells were fixed and labeled with anti-NF- B (p65) antibody and an FITC-conjugated secondary antibody. Individual cells were imaged as described in MATERIALS AND METHODS. One of three similar experiments is shown. PDTC, pyrrolidine dithiocarbamate.
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It has been shown that cell activation by cytokines leads to the degradation of I
B-
, accompanied by NF-
B translocation to the nucleus. To determine whether IL-1
stimulated degradation of I
B-
and NF-
B translocation, the cells were stimulated by 1.5 ng/ml of IL-1
for various times. The cytosolic and nuclear fractions were used to determine the degradation of I
B-
and NF-
B translocation by Western blotting analysis using anti-I
B-
or anti-NF-
B (p65), respectively. As shown in Fig. 5B, IL-1
rapidly stimulated activation of IL-1
and degradation of I
B-
in 10 min that reached a maximal response within 30 min, and returned to the basal level within 60 min during the period of observation. In contrast, IL-1
stimulated translocation of NF-
B (p65) into the nucleus within 10 min, which was sustained over 60 min and slightly declined.
p42/p44 MAPK, p38, JNK phosphorylation, and NF-
B are necessary for the VCAM-1 protein expression in HTSMC induced by IL-1
; therefore, it is important to determine whether these MAPK phosphorylations are associated with NF-
B activation. To examine this possibility, activation of NF-
B was assessed following IL-1
stimulation in the presence of inhibitors for MEK1/2, p38, JNK, and helenalin, respectively. IL-1
-stimulated time-dependent activation of NF-
B and degradation of I
B-
was significantly inhibited by preincubation with helenalin (P < 0.05, n = 3, compared with cells exposed to IL-1
alone), but not by U0126, SB-202190, and SP-600125 (Fig. 5B). Correspondingly, the images of immunofluorescence staining showed that IL-1
-induced NF-
B translocation was blocked with pretreatment of helenalin, but not by the pharmacological inhibitors of these MAPKs (Fig. 5C). These results indicated that IL-1
-stimulated NF-
B translocation is essential for VCAM-1 upregulation and is independent on activation of p42/p44 MAPK, p38 MAPK, and JNK in HTSMC.
MAPKs and NF-
B are required for VCAM-1 mRNA expression induced by IL-1
.
We further examined whether these kinases were involved in regulation of VCAM-1 expression at the level of transcription in these cells. As shown in Fig. 6, pretreatment of HTSMC with inhibitors of MEK1/2 (U0126 and PD-98059), p38 (SB-202190), JNK (SP-600125), and NF-
B (helenalin) significantly attenuated IL-1
-induced VCAM-1 mRNA accumulation assessed by RT-PCR. These results further indicated that in HTSMC, regulation of VCAM-1 expression through activation of p42/p44 MAPK, p38, JNK, and NF-
B occurs mainly at the gene transcriptional level.
Adhesion of neutrophils on HTSMC treated with IL-1
.
IL-1
has been recognized as a potent proinflammatory mediator that increases the expression of adhesion molecules (2, 5, 12) and the adhesiveness between leukocytes and several cell types (11, 12, 19). To test the functional activity of VCAM-1 expressed on HTSMC, we assessed the ability of purified PMN to adhere to IL-1
-stimulated HTSMC. As shown in Fig. 7, the value of PMN adhesion to the HTSMC monolayer was significantly increased (
6-fold) by stimulation with 0.15 ng/ml of IL-1
for 4 h (P < 0.05, n = 3, compared with the basal level). This enhanced adhesion was attenuated by pretreatment of HTSMC with 1 µM U0126, 10 µM SP-600125, 30 µM SB-202190, or 10 µM helenalin before exposure to IL-1
. To further determine the surface molecules responsible for PMN adhesion to HTSMC monolayers, PMN adhesion was assessed in the presence of antibodies to VCAM-1 and ICAM-1. PMN adhesion to IL-1
-stimulated HTSMC monolayers was significantly inhibited by preincubation with these adhesion molecule antibodies.
 |
DISCUSSION
|
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Upregulation of adhesion molecules on the surface of the airway epithelium may play a key role in recruitment and infiltration of PMN at sites of inflammation in airways (19, 27, 36). IL-1
has been shown to be one of the major inducers of ICAM-1 in both in vivo and in vitro models in the airways (5, 12). IL-1
has also been demonstrated to induce the expression of VCAM-1 in lung epithelial cells, but little is known about the intracellular signaling pathways leading to its expression in lung cells, except NF-
B (12). IL-1
has been shown to activate all of these three MAPK pathways in several cell types (20, 47). It has been well established that p38 and JNK mediate signals stimulated by IL-1
, heat shock, osmotic shock, ultraviolet light, and DNA-damaging agents, whereas activation of p42/p44 MAPK plays an important role in mediating cell proliferation in response to growth factors and mitogens (17, 43, 55). Because IL-1
induces a wide range of different actions, the phosphorylation of these three MAPKs (20, 47) may not directly imply an involvement of these three MAPKs in the IL-1
-induced VCAM-1 expression and protein production. Moreover, there is no report that suggests activation of p42/p44 MAPK or others is required for IL-1
-induced VCAM-1 expression in HTSMC. Therefore, the role of MAPKs involved in IL-1
-induced VCAM-1 gene expression and protein production was investigated by using the specific pharmacological inhibitors of these MAPKs.
In this study, we investigated IL-1
-induced signaling cascades that lead to increased expression of VCAM-1 in HTSMC. The IL-1
-induced VCAM-1 expression was significantly attenuated by U0126 and PD-98059 (inhibitors of MEK1/2), SB-202190 (inhibitor of p38), SP-600125 (an inhibitor of JNK), and helenalin (an inhibitor of NF-
B). Furthermore, activation of NF-
B was inhibited by helenalin, but not by U0126, SB-202190, and SP-600125. Our results establish mechanisms underlying the activation of p42/p44 MAPK, p38, and JNK as well as the activation of NF-
B, a transcription factor, by IL-1
that may be critical in the upregulation of VCAM-1. The involvement of NF-
B in the induction of VCAM-1 expression by IL-1
likely results from the degradation of I
B-
by IL-1
and in turn leads to activation of NF-
B in HTSMC.
IL-1
has been shown to activate p42/p44 MAPK in a variety of cell types. However, there is no report suggesting that activation of p42/p44 MAPK is required for IL-1
-induced VCAM-1 expression. In this study, our results provide the first evidence that activation of p42/p44 MAPK is necessary for IL-1
-induced VCAM-1 mRNA expression and protein production in HTSMC. Both U0126, a specific inhibitor of MEK1/2 (18), and PD-98059 (15) also blocked IL-1
-induced VCAM-1 mRNA expression and protein production in a concentration-dependent manner in HTSMC. At the highest concentrations used, U0126 (1 µM) and PD-98059 (10 µM) almost completely inhibited IL-1
-induced VCAM-1 expression. U0126 also attenuated the IL-1
-stimulated p42/p44 MAPK phosphorylation. These results are consistent with those showing IL-1
-induced expression of other genes, such as COX-2 (53), chemokine (7), and ICAM-1 (2), in which activation of p42/p44 MAPK is necessary. In contrast, IL-1
-induced adhesion molecule expression was not involved in p42/p44 MAPK activation. In that study, PD-98059 has been shown to inhibit p42/p44 MAPK activation but had no effect on ICAM-1 expression in response to IL-1
.
The involvement of p38 MAPK in IL-1
-induced VCAM-1 expression was also investigated using SB-202190, which is a specific inhibitor of p38 MAPK (24). In HTSMC, SB-202190 specifically blocked the activation of p38 MAPK induced by IL-1
and led to a significant decrease in VCAM-1 mRNA expression and protein production. These results indicate that p38 MAPK pathway was involved in the IL-1
-induced VCAM-1 expression, consistent with the idea that p38 MAPK activation is required for the expression of both VCAM-1 and ICAM-1 in cardiac cells (25). This is confirmed by the results that activation of p38 MAPK is also necessary for IL-1
-induced chemokine expression (7) and COX-2 expression in HTSMC (53) and monocyte chemoattractant protein-1 expression in human mesangial cells (45).
In addition to activation of p42/p44 MAPK and p38 MAPK, IL-1
-stimulated activation of JNK has also been demonstrated in several cell types (20, 47). Next, we investigated the involvement of JNK in IL-1
-induced VCAM-1 expression using a novel selective JNK inhibitor, SP-600125 (6). Pretreatment of HTSMC with SP-600125 attenuated IL-1
-induced VCAM-1 expression in a concentration-dependent manner, suggesting that VCAM-1 induction is mediated through a JNK-dependent mechanism in HTSMC. We also found a sustained decrease of the IL-1
-induced JNK phosphorylation by SP-600125 at a concentration of 0.1 µM. Our results are also consistent with reports that activation of JNK is essential for the upregulation of ICAM-1 and VCAM-1 (13, 34, 42, 48).
It is worth noting that our results are in contrast to those obtained in the A549 cells treated with IL-1
at a concentration of 1 ng/ml (12). Under those conditions, Chen et al. (12) excluded the involvement of p42/p44 MAPK, p38, and JNK in the IL-1
-induced expression of ICAM-1, using specific kinase inhibitors, although PD-98059 and SB-203580 almost completely inhibited the phosphorylation of p42/p44 MAPK and p38 but had no effect on ICAM-1 expression in response to IL-1
. This evident difference between the report by Chen et al. (12) and our study may be due to the different cells used. Similarly, in our study, all of these three kinase inhibitors had no effect to block the IL-1
-induced expression of ICAM-1 when a concentration of IL-1
>1 ng/ml was used (data not shown). Exposure of HTSMC to IL-1
even at very low concentrations (i.e., 1.5 pg/ml) still induced a large amount of ICAM-1 expression that was not susceptible to most inhibitors used to block VCAM-1 expression in this study. In addition, we speculate that both ICAM-1 and VCAM-1 expression induced by IL-1
might be mediated through a similar mechanism. This is the reason why measurement of VCAM-1 was used as a parameter for IL-1
-induced adhesion molecule expression in this study. The degree to which IL-1
-induced adhesion molecule expression was inhibited seemed to be dependent on the extents of MAPK phosphorylation. Because IL-1
is known to activate several signaling transduction pathways (20, 47), it is possible that treatment with one of these three kinase inhibitors reduced only one of several components necessary for, or involved in, IL-1
-induced VCAM-1 expression. A balance may also exist among these pathways leading to IL-1
induction of VCAM-1 expression. For example, dependence of different MAPK pathways on VCAM-1 expression may have predominated and reflected on the potency of a specific inhibitor when HTSMC were exposed to a low concentration of IL-1
, such as 1.5 pg/ml in this study, but this inhibitory effect may be overcome with increasing concentrations of IL-1
, since other signaling pathways may now dominate to be sufficient for VCAM-1 expression. This explanation may also, in part, account for why MAPK inhibitors do not completely abolish IL-1
-induced VCAM-1 expression.
It has been well established that inflammatory responses following exposure to cytokines are highly dependent on activation of NF-
B transcription factor, which plays an important role in regulation of several gene expressions (7, 12, 20, 39). The sequestration of NF-
B by I
B in the cytoplasm and I
B kinase phosphorylation leading to proteasomal degradation of I
B-
, resulting in activation and translocation of NF-
B into nucleus, is essential in expression of several genes, such as ICAM-1 in A549 cells (12). In this study, VCAM-1 expression induced by IL-1
was completely abolished by the specific NF-
B inhibitors helenalin and PDTC, indicating that activation of NF-
B is involved in IL-1
-induced expression of VCAM-1. The increase in NF-
B translocation correlates with the rapid and transient degradation of I
B-
in the cytosol of HTSMC treated with IL-1
. Our results suggest that degradation of I
B-
may initiate translocation of active NF-
B into the nucleus. It should be noted that IL-1
induced a rapid degradation of I
B-
within 10 min. Afterward, its level was restored close to that of unstimulated cells. This may be due to the fact that the I
B-
gene itself has a
B site in its promoter region, so that NF-
B induces its synthesis (4). However, NF-
B sustained to be activated within 60 min. Moreover, the rapid activation of NF-
B following IL-1
exposure was observed, which was also inhibited by helenalin. These results are consistent with those of the report by Chen et al. (12) in which IL-1
-induced expression of VCAM-1 was mediated through NF-
B activation. Interestingly, activation of p42/p44 MAPK, p38, and JNK as well as NF-
B appears to be involved in IL-1
-induced expression of VCAM-1. However, it remains unclear how the activation of p42/p44 MAPK, p38, and JNK is associated with VCAM-1 gene expression. Previously, it has been established that MEKK1 induces activation of both IKK-
and IKK-
, leading to NF-
B activation (4, 20). Thus p42/p44 MAPK activation may be required for NF-
B activation after exposure to IL-1
. In this study, our results demonstrated that IL-1
-stimulated NF-
B activation was not significantly inhibited by U0126, SB-202190, and SP-600125, indicating that both MAPK cascades and NF-
B independently regulated gene expression of VCAM-1 in HTSMC. On the basis of results obtained in this study, IL-1
induces NF-
B nuclear translocation independently of these MAPK pathways. These results suggest that the activation of NF-
B and the activation of p42/p44 MAPK, p38, and JNK are mediated by distinct pathways (52) or these MAPK pathways may converge at a step involving NF-
B transactivation (50, 52). How the VCAM-1 gene is regulated by IL-1
through p42/p44 MAPK, p38 MAPK, and JNK remains to be investigated. In addition, NF-
B binding sites have been identified previously in the ICAM-1 gene promoter (12), which might explain the modulation exerted by IL-1
through NF-
B activation.
To our knowledge, this study is the first in which the involvement of p42/p44 MAPK, p38 MAPK, and JNK, as well as NF-
B in the IL-1
-induced expression of VCAM-1, is observed in HTSMC. The present study provides evidence that activation of the p42/p44 MAPK pathway by IL-1
is necessary for VCAM-1 expression in HTSMC. In addition to p42/p44 MAPK, p38 MAPK and JNK have been shown to be additional MAPKs required for IL-1
-induced VCAM-1 expression in these cells. We also found that IL-1
-induced VCAM-1 expression is mediated by the NF-
B pathway based on the early NF-
B translocation and suppression of VCAM-1 expression in the presence of the specific NF-
B inhibitors helenalin and PDTC. The mechanisms by which IL-1
induces VCAM-1 expression by HTSMC may be an important link in the pathogenesis of airway inflammatory diseases. Therefore, understanding the mechanisms underlying IL-1
-induced VCAM-1 expression in HTSMC is important in the development of new therapeutic strategies.
 |
GRANTS
|
---|
This work was supported by Chang Gung Medical Research Foundation Grant CMRPD32043 and National Science Council, Taiwan, Grant NSC92-2320-182-020.
 |
ACKNOWLEDGMENTS
|
---|
The authors thank H. J. Chung and L. D. Hsiao for technical assistance in the preparation of the manuscript and art work of this study.
 |
FOOTNOTES
|
---|
Address for reprint requests and other correspondence: C.-M. Yang, Dept. of Pharmacology, Chang Gung Univ., 259 Wen-Hwa 1st Road, Kwei-San, Tao-Yuan, Taiwan (E-mail: chuenmao{at}mail.cgu.edu.tw)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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