AJP - Lung Journal of Neurophysiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Lung Cell Mol Physiol 291: L84-L90, 2006. First published January 27, 2006; doi:10.1152/ajplung.00388.2005
1040-0605/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
291/1/L84    most recent
00388.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (7)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lee, S. Y.
Right arrow Articles by Shim, J. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lee, S. Y.
Right arrow Articles by Shim, J. J.

Peroxisome proliferator-activated receptor-{gamma} inhibits cigarette smoke solution-induced mucin production in human airway epithelial (NCI-H292) cells

Sung Yong Lee,1 Eun Joo Kang,1 Gyu Young Hur,1 Ki Hwan Jung,3 Hye Cheol Jung,2 Sang Yeub Lee,2 Je Hyeong Kim,3 Chol Shin,3 Kwang Ho In,2 Kyung Ho Kang,1 Se Hwa Yoo,2 and Jae Jeong Shim1

1Department of Internal Medicine, Guro Hospital, Korea University, Seoul; 2Department of Internal Medicine, Anam Hospital, Korea University, Seoul; and 3Department of Internal Medicine, Ansan Hospital, Korea University, Ansan, Korea

Submitted 9 September 2005 ; accepted in final form 16 January 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The main etiologic factor for chronic bronchitis is cigarette smoke. Exposure to cigarette smoke is reported to induce goblet cell hyperplasia and mucus production. Mucin synthesis in airways has been reported to be regulated by the EGFR system. Peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) is a member of the ligand-activated nuclear receptor superfamily. PPAR-{gamma} is implicated in anti-inflammatory responses, but mechanisms underlying these varied roles remain ill-defined. Recently, reports have shown that upregulation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) might be one of the mechanisms through which PPAR-{gamma} agonists exert their anti-inflammatory actions. However, no data are available on the role of PPAR-{gamma} in smoke-induced mucin production. In this study, we investigated the effect of PPAR-{gamma} agonist (rosiglitazone) on smoke-induced mucin production in NCI-H292 cells. Exposure to cigarette smoke causes a significant decrease in PTEN expression and increases dose-dependent EGFR-specific tyrosine phosphorylation, resulting in MUC5AC mucin production in NCI-H292 cells. PPAR-{gamma} agonists or specific inhibitors of phosphoinositide 3-kinase exert inhibition of cigarette smoke-induced mucin production, with the upregulation of PTEN signaling and downregulation of Akt expression. This study demonstrates that PPAR-{gamma} agonist functions as a regulator of epithelial cell inflammation that may result in reduction of mucin-producing cells in airway epithelium.

rosiglitazone; epidermal growth factor receptor; phosphatase and tensin homolog deleted on chromosome 10; smoking

THE CLINICAL HALLMARKS of chronic bronchitis are cough and excessive mucus production (40, 46). The main etiologic factor for chronic bronchitis is cigarette smoke. Exposure to cigarette smoke is reported to induce goblet cell hyperplasia and mucus production in several species (24, 33, 46). Mucin synthesis in airways has been reported to be regulated by the epidermal growth factor receptor (EGFR) system: activation of EGFR tyrosine kinase by its ligands leads to the synthesis of mucin MUC5AC at both mRNA and protein levels in airway epithelial cells in vitro and causes mucin MUC5AC production and goblet cell metaplasia in vivo (6, 15, 29, 33, 41, 42, 44, 45). Cigarette smoke can upregulate the EGFR mRNA expression and induce activation of EGFR-specific tyrosine phosphorylation, resulting in upregulation of MUC5AC mRNA and protein production through downstream signaling of EGFR including the Akt/phosphoinositide 3-kinase (PI3K) and Ras/MEK/MAPK cascade (33, 35, 41, 46). Peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) is a member of the ligand-activated nuclear receptor superfamily. PPAR-{gamma} has been shown to regulate differentiation and/or cell growth in many cell types. Moreover, PPAR-{gamma} is also implicated in anti-inflammatory responses, but mechanisms underlying these varied roles remain ill-defined (23). Recently, reports show that upregulation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), with the concomitant downregulation of PI3K-dependent signaling pathways (19, 39, 50), might be one of the mechanisms through which PPAR-{gamma} agonists exert their anti-inflammatory actions. However, no data are available on the role of PTEN and PPAR-{gamma} in mucin production.

In the present study, we investigated the effects of PPAR-{gamma} agonist (rosiglitazone) on PTEN expression and smoke-induced mucin production in NCI-H292 cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Preparation of cigarette smoke solution. Standard research cigarettes (code 2R1, produced by the University of Kentucky Tobacco and Health Research Foundation) were used in this study. Cigarette smoke solution was prepared as described previously (46). In brief, cigarette smoke was withdrawn into a polypropylene syringe (50 ml) at a rate of one puff/min and bubbled slowly into 50 ml of RPMI 1640 medium containing 50 mM HEPES buffer. The smoke solution was then titrated to pH 7.4 and used immediately after preparation.

Cell culture. NCI-H292 cells, a human pulmonary mucoepidermoid carcinoma cell line, were grown in RPMI 1640 containing 10% fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin (100 µg/ml), and HEPES (25 mM) at 37°C in a humidified 5% CO2 water-jacketed incubator. Six-well culture plates were used to culture the cells. When confluent, cells were incubated for 1 h with cigarette smoke solution. The cells were then washed and incubated with fresh medium alone. Experiments were terminated at preselected times (for protein, 24 h). For the controls, cells were incubated with medium alone for the same time periods. In inhibition studies, NCI-H292 cells were pretreated with wortmannin (20 nM; Sigma Aldrich, St. Louis, MO), LY-294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one, 30 µM; Sigma Aldrich], and tyrphostin AG-1478 (10 µM, Calbiochem) 30 min before the cigarette smoke solution was delivered. To evaluate the effect of PPAR-{gamma} on smoke-induced mucin production, cells were pretreated with PPAR-{gamma} agonist (rosiglitazone) 30 min before the cigarette smoke solution was delivered.

Measurement of TNF-{alpha}. NCI-H292 cells were incubated with cigarette smoke solution or were treated in culture medium with various concentrations of rosiglitazone (0.1, 1 µM) 30 min before they were incubated in cigarette smoke solution. The TNF-{alpha} level was determined by ELISA (Sigma) from supernatants harvested at 24 h.

Immunoassay of MUC5AC protein. NCI-H292 cells were incubated with cigarette smoke solution or were treated in culture medium with wortmannin, LY-294002, AG-1478, rosiglitazone, or TNF-{alpha} monoclonal antibody (infliximab). Infliximab was purchased from a pharmaceutical supplier (Schering Korea, Seoul, Korea). MUC5AC protein was measured. In brief, cell culture supernatants were prepared with PBS at multiple dilutions, and 50 µl of each sample were then incubated with bicarbonate-carbonate buffer (50 µl) at 40°C in a 96-well plate (Maxisorp Nunc; Fisher Scientific, Santa Clara, CA) until dry. The plates were then rewashed three times with PBS and blocked with 2% bovine serum albumin, fraction V (Sigma Aldrich) for 1 h at room temperature. The plates were then rewashed three times with PBS and incubated with 50 µl of MUC5AC monoclonal antibodies (clone 45 M1, 1:100; NeoMarkers, Fremont, CA) diluted with PBS containing 0.05% Tween 20. After 1 h, the wells were washed three times with PBS, and 100 µl of horseradish peroxidase-goat anti-mouse IgG conjugate (1:10,000) were dispensed into each well. After a further 1 h, the plates were washed three times with PBS. Color reaction was developed using 3,3',5,5'-tetramethylbenzidine peroxidase solution (Kirkegaard and Perry Laboratories, Gaithersburg, MD) and stopped with 2 N H2SO4. Absorbance was read at 450 nm. Each experiment was repeated at least five times. Mucin of the submandibular gland was used as a positive ELISA control and naïve medium (RPMI+10% FBS, never exposed to cells) was used as a negative control.

Western blotting. NCI-H292 cells were harvested and washed twice with PBS. The cell pellet was lysed with mammalian protein extraction reagent lysis buffer (Pierce, Rockford, IL) and 1:100 dilution of proteinase inhibitors (Pierce). To remove insoluble materials, cell lysates were centrifuged at 14,000 rpm for 5 min, and 1 vol of Laemmli sample buffer (4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 4 mg/100 ml bromphenol blue, and 125 mM Tris·HCl, pH 6.8; Bio-Rad Laboratories) was added to the supernatant. After incubation at 95°C for 5 min, total proteins were separated by SDS-PAGE in 10% acrylamide gels. The running gel was electro-transferred to polyvinylidene difluoride membrane (Bio-Rad Laboratories), which was blocked with 5% fat-free milk in Tris-buffered saline/0.05% Tween 20 for 1 h at room temperature. Membranes were incubated with anti-EGFR (Ab-2) monoclonal antibody (EGFR, 1:1,000, 100 µg/ml; Oncogene), anti-phospho-specific EGFR monoclonal antibodies (EGFR-p, 1:1,000, 1 µg/ml; Calbiochem), anti-PTEN (1:800, Santa Cruz Biotechnology), and anti-pAkt (1:1,000, Calbiochem). The membranes were stripped and reblotted with monoclonal anti-beta-actin antibody (Sigma Aldrich) to verify equal loading of protein in each lane. The membranes were incubated with horseradish peroxidase-linked secondary antibody (Vector, Burlingame, CA) for 1 h at room temperature. Proteins were detected using the Supersignal West picochemiluminescent substrate kit (Pierce, Rockford, IL) according to the manufacturer's instructions.

Statistics. All data are expressed as means ± SE. One-way analysis of variance (ANOVA) was used to determine statistically significant differences between groups. Scheffé's F-test was used to correct for multiple comparisons when statistical significances were identified in the ANOVA. P < 0.05 for the null hypothesis was accepted as indicating a statistically significant difference.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Cigarette smoke induces EGFR tyrosine phosphorylation and MUC5AC protein synthesis. Because the activation of EGFR leads to MUC5AC synthesis (45), we examined the effect of cigarette smoke solution on activation of the EGFR tyrosine kinase, and AG-1478, a potent and selective chemical inhibitor of EGFR tyrosine kinase activity, was used to block EGFR signal transduction. As expected, pretreatment (30 min) with cigarette smoke solution induced EGFR-specific tyrosine phosphorylation, and AG-1478 inhibited the cigarette smoke solution-induced increase of p-EGFR levels (Fig. 1). The concentrations were determined from previous studies (47). NCI-H292 cells were treated with various doses of cigarette smoke. Cigarette smoke solution caused increase in MUC5AC protein synthesis dose dependently (Fig. 2).


Figure 1
View larger version (53K):
[in this window]
[in a new window]
  		Fig. 1. Tyrosine phosphorylation of epidermal growth factor receptor (EGFR) induced by cigarette smoke was inhibited by EGFR tyrosine kinase inhibitor (AG-1478). The cells were pretreated with or without AG-1478 (10 µM) for 30 min and then treated with vehicle or cigarette smoke solution for 1 h. After lysis, EGFR was immunoprecipitated with antiphosphorylated EGFR antibody (top) and with anti-EGFR antibody (bottom). EGFR expression was not significantly different for the different lysates, thus indicating equal protein loading. p, Phosphorylated.

 

Figure 2
View larger version (8K):
[in this window]
[in a new window]
  		Fig. 2. Effects of cigarette smoke solution on MUC5AC production in NCI-H292 cells. Cigarette smoke was withdrawn into a polypropylene syringe (50 ml) at a rate of 1 puff/min and then bubbled slowly into RPMI 1640 containing 50 mM HEPES buffer. Cells were incubated for 1 h in cigarette smoke solutions. They were then washed and incubated with fresh medium alone. MUC5AC levels were determined in the supernatants harvested after 24 h by ELISA. MUC5AC protein was calculated as percentage with respect to control. Cigarette smoke solution increased MUC5AC production dose dependently (n = 5, *P < 0.05, **P < 0.01 compared with control).

 
Rosiglitazone inhibits cigarette smoke-induced TNF-{alpha} production from NCI-H292 cells. TNF-{alpha} can induce EGFR expression, resulting in MUC5AC production as noted in previous studies (45). For the role of TNF-{alpha} in cigarette smoke-induced mucin production, we used a TNF-{alpha} monoclonal antibody (infliximab) that selectively blocks the effects of TNF-{alpha} and measured MUC5AC production. Cigarette smoke-induced MUC5AC protein synthesis was inhibited by infliximab dose dependently (Fig. 3). Therefore, we measured TNF-{alpha} levels in cigarette smoke-stimulated NCI-H292 cells. NCI-H292 cells were treated with cigarette smoke solution and rosiglitazone. TNF-{alpha} levels were measured in the culture supernatants of the cells. Cigarette smoke solution increased TNF-{alpha} production in NCI-H292 cells, whereas rosiglitazone had an inhibitory effect on TNF-{alpha} production in cigarette smoke-stimulated NCI-H292 cells in a dose-dependent fashion (Fig. 4).


Figure 3
View larger version (10K):
[in this window]
[in a new window]
  		Fig. 3. Effects of TNF-{alpha} inhibitor (infliximab) on cigarette smoking solution-induced MUC5AC production in NCI-H292 cells. The cells were pretreated with or without infliximab for 30 min and then treated with cigarette smoke solution for 1 h. MUC5AC levels were determined in the supernatants harvested after 24 h by ELISA. MUC5AC protein was calculated as percentage with respect to control. Cigarette smoke solution-induced MUC5AC protein synthesis was inhibited by TNF-{alpha} monoclonal antibody (infliximab) dose dependently (n = 5, *P < 0.05, **P < 0.01 compared with 9 puffs of cigarette smoke solution alone).

 

Figure 4
View larger version (7K):
[in this window]
[in a new window]
  		Fig. 4. Cigarette smoke-induced TNF-{alpha} secretion was inhibited by rosiglitazone dose dependently in NCI-H292 cells. The cells were pretreated with or without rosiglitazone (0.1 µM/1 µM) for 30 min and then stimulated with vehicle or cigarette smoke solution for 1 h. We used ELISA to generate data, which are expressed as mean values ± SE (n = 5, *P < 0.01 compared with control, **P < 0.05, {dagger}P < 0.01 compared with 9 puffs of cigarette smoke solution alone).

 
Effect of rosiglitazone, AG-1478, and PI3K inhibitors on MUC5AC production in NCI-H292 cells. To test whether the cigarette smoke-induced MUC5AC production is related to the EGFR and Akt/PI3K pathway, cells were incubated with EGFR tyrosine kinase inhibitor, selective PI3K inhibitors (wortmannin, LY-294002), and rosiglitazone. Cigarette smoke-induced MUC5AC synthesis was inhibited by pretreatment with EGFR tyrosine kinase inhibitor and selective PI3K inhibitors. Furthermore, pretreatment of the cells with PPAR-{gamma} agonist (rosiglitazone) prevented MUC5AC protein synthesis, in a dose-dependent fashion (Fig. 5). These results indicate that smoke-induced MUC5AC mucin production is related, at least in part, to the Akt/PI3K pathway.


Figure 5
View larger version (11K):
[in this window]
[in a new window]
  		Fig. 5. Effects of Akt inhibitor, EGFR inhibitor, or rosiglitazone on cigarette smoking solution induced MUC5AC production in NCI-H292 cells. The cells were pretreated with wortmannin (20 nM), LY-294002 (30 µM), or AG-1478 (10 µM) 30 min before the cigarette smoke solution was delivered and then stimulated with vehicle or cigarette smoke solution for 1 h. MUC5AC levels were determined in the supernatants harvested after 24 h by ELISA. MUC5AC protein was calculated as percentage with respect to control (n = 5, *P < 0.05 compared with cigarette smoke alone, {dagger}P < 0.05 compared with rosiglitazone 0.1 µM).

 
Rosiglitazone increases PTEN expression with concomitant downregulation of pAkt expression. Because rosiglitazone inhibited the MUC5AC mucin production, we examined whether inhibited mucin production is related to PTEN expression. PTEN, which antagonizes the action of PI3K by dephosphorylation of phosphatidylinositol 3,4,5-triphosphate (PIP3) has been suggested as a possible mechanism of anti-inflammatory action of PPAR-{gamma} agonist (37). Pretreatment of NCI-H292 cells with rosiglitazone increased PTEN expression, which was reduced by cigarette smoke solution. We examined activated Akt (p-Akt), which cannot activate the subsignal pathway while under the PTEN activation state. Cigarette smoke-induced activated Akt expression was inhibited by pretreatment with rosiglitazone (Fig. 6).


Figure 6
View larger version (33K):
[in this window]
[in a new window]
  		Fig. 6. Effects of smoking and rosiglitazone on the expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and pAkt in NCI-H292 cells. The cells were pretreated with or without rosiglitazone (0.1 µM/1 µM) for 30 min and then stimulated with vehicle or cigarette smoke solution for 1 h. Western blot analysis for PTEN and phospho (p) Akt were measured at 24 h after smoking solution treatment. Sample loading was normalized using a probe for beta-actin. Cigarette smoke solution downregulated PTEN expression with concomitant upregulation of pAkt signaling. However, rosiglitazone upregulated PTEN expression with concomitant downregulation of pAkt signaling. Data shown are representative of 2 independent experiments/treatments.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Our study examined the effect of PPAR-{gamma} agonist (rosiglitazone) on mucin production in airway epithelium by using NCI-H292 cells. Delivery of the rosiglitazone to cigarette smoke-induced NCI-H292 cells decreased MUC5AC protein expression with increased PTEN expression. As a result of such increases in the expression of PTEN, the PI3K/Akt pathway was inhibited, and inhibition of Akt pathway abrogated MUC5AC mucin production. To our knowledge, this report is the first to describe this effect of rosiglitazone on cigarette smoke-induced mucin production.

Chronic bronchitis remains one of the most common chronic airway inflammatory diseases and is associated with epithelial metaplasia and mucus production. Various mediators, such as transforming growth factor (TGF)-beta, granulocyte-macrophage colony-stimulating factor, EGF, and TNF-{alpha} have been reported to regulate airway inflammation. Exposure to cigarette smoke activates an inflammatory cascade in the airway epithelium, resulting in the production of a number of potent cytokines and chemokines, with accompanying damage to the lung epithelium, increased permeability, and recruitment of macrophages and neutrophils to the airway (1). Upregulation of cytokine gene expression in epithelial cells has been linked to the activation of specific signaling pathways. EGFR activation also plays a major role in modulating mucus production in airway epithelium. Takeyama et al. (46) reported that exposure to cigarette smoke upregulated EGFR mRNA expression and induced EGFR-specific tyrosine phosphorylation, resulting in upregulation of MUC5AC mucin mRNA and protein production. EGFR activation may involve two pathways, namely, ligand-dependent and ligand-independent EGFR tyrosine phosphorylation (6). In ligand-dependent EGFR tyrosine phosphorylation, EGFR ligands (EGF or TGF-{alpha}) bind to EGFRs in the extracellular domain and activate them, whereas, in ligand-independent EGFR tyrosine phosphorylation, EGFR tyrosine phosphorylation occurs in the absence of exogenous EGFR ligands. Ligand-independent EGFR phosphorylation is reported in response to oxidative stress that can be produced by cigarette smoke and by activated neutrophils (20, 44, 46).Upon activation, these receptors undergo dimerization and tyrosine autophosphorylation, followed by recruitment and activation of signaling molecules that contain MAPK and Akt, resulting in airway epithelial proliferation and mucus hypersecretion (3, 6, 48). Previous studies reported that the MAPK pathway is mainly involved in mucin production induced by a variety of stimuli (15). However, in recent years, a second pathway, downstream of PI3K and Akt, has become a focus of interest (3, 48). The activated PI3K converts the plasma membrane lipid phosphatidylinositol-4,5-biphosphate to phosphatidylinositol-3,4,5-triphosphate (9). This phosphorylation then stimulates the catalytic activity of Akt. Active Akt phosphorylates I{kappa}B kinase-{alpha} (IKK{alpha}), which promotes activation of its heterodimeric partner, IKKbeta, in the IKK{alpha}/IKKbeta complex (25, 36). The IKK complex phosphorylates I{kappa}B, thereby promoting its dissociation from NF-{kappa}B (5). This process allows NF-{kappa}B to enter the nucleus, where it activates cytokines, adhesion molecules and other target genes (10). NF-{kappa}B has been shown to play an important role in mucus production (11, 31). Thus PI3K/Akt signaling may render cells produce mucin. In the present study, our results show that exposure of airway epithelial cells to cigarette smoke activates EGFR tyrosine phosphorylation, causing MUC5AC mucin synthesis in NCI-H292 cells (Figs. 1 and 2). The Akt phosphorylation is increased, while the PTEN protein expression is reduced, and Akt pathway inhibitor LY-294002 suppressed MUC5AC production in cigarette smoke solution-treated NCI-H292 cells.

PPARs have been reported to regulate inflammatory responses (13). Three subtypes of PPARs ({alpha}, beta, and {gamma}) have been identified (16). One of the natural ligands for PPAR-{gamma} is 15-deoxy-{Delta}-prostaglandin J2, the major metabolite of prostaglandin D2 (17). Ligand-induced activation of PPAR results in heterodimerization of the receptor with the retinoid X receptor (RXR) and subsequent binding to specific peroxisome proliferator-responsive elements located within the promoter region of target genes (27). PPAR-{gamma} has been shown to play a major role in regulating adipocyte differentiation and glucose homeostasis. One group of synthetic PPAR-{gamma}-agonistic ligands is rosiglitazone, a group of drugs used in the treatment of diabetic patients (49). In addition, there is a growing body of evidence that suggests that PPAR-{gamma} agonists may exert anti-inflammatory action in vivo (2, 30, 34), and some reports have demonstrated that ligand-activated PPAR-{gamma} can downregulate NF-{kappa}B transcription (12, 38). Therefore, inhibition of NF-{kappa}B activation by PPAR-{gamma} agonist may well reduced expression of proinflammatory mediators (TNF-{alpha}, ICAM-1, etc.). A possible clue to the mechanism of PPAR-{gamma} anti-inflammatory action has been provided by the observation that expression of PTEN, which antagonizes PI3K-mediated signaling, is directly upregulated by PPAR-{gamma} (37). PPAR-{gamma} is able to bind two PPAR response elements in genomic sequence upstream of PTEN, and antisense-mediated disruption of PPAR-{gamma} expression prevented the upregulation of PTEN. The PTEN gene constitutes a novel candidate tumor suppressor located on chromosomal band 10q23.3 and exists as a 55-kDa protein comprising an NH2-terminal catalytic domain and a dual-specificity phosphatase (32). It is known that PTEN plays critical roles not only in suppressing cancer but also in embryonic development, cell migration, cell signaling, and apoptosis (4, 14, 21, 43). PTEN appears to serve as a hub or switch point that links complex signaling pathways. Expression of PTEN has opposite effects, such as blocking downstream signaling of EGFR including the Ras/MEK/MAPK cascade and PI3K/Akt signaling (21, 22, 28, 51). The activation of PTEN function results in a decrease of PIP3 concentration, which in turn leads to Akt inactivation. This would appear to suggest that the anti-inflammatory function of PTEN is exerted through the negative regulation of the PI3K/Akt cell signaling pathway (43). Our results show that administration of rosiglitazone results in increase of PTEN expression. The Akt phosphorylation that is increased by cigarette smoking solution is significantly reduced by the administration of rosiglitazone. Together, these findings suggest that PPAR-{gamma} agonist may reduce smoking solution-induced MUC5AC synthesis by increasing PTEN expression.

In conclusion, in our study of cigarette smoke-induced airway epithelial cells exposed to PPAR-{gamma} agonist, decreased inflammatory cytokine (TNF-{alpha}) and MUC5AC mucin synthesis was observed, which was shown to be associated with the inhibition of PI3K/Akt pathway (Fig. 7). These findings thus represent the first evidence for a PPAR-{gamma}-dependent and therapeutically significant inhibition of cigarette smoke-induced mucin production. Therefore, activation of PTEN and inhibition of PI3K/Akt by PPAR-{gamma} agonist are proposed as a potential therapy for cigarette smoke-induced mucin secretory diseases. Most patients with chronic obstructive pulmonary disease (COPD) have mucus hypersecretion. Overproduced mucus plugs the airways and contributes to exacerbations of COPD. Presently, there is no effective therapy to relieve the symptoms and to halt the progression of this disease. Our results provide a strategy for therapy by recovery of PTEN expression.


Figure 7
View larger version (18K):
[in this window]
[in a new window]
  		Fig. 7. Model for the inhibition of MUC5AC by rosiglitazone. This model combines others' previous results on mechanisms responsible for the ability of smoke to activate EGFR with the present results showing mechanisms responsible for MUC5AC inhibition. TNF serves as an initial stimulus, which results in transactivation of EGFR, through mechanisms that are dependent on tumor necrosis factor-{alpha}-converting enzyme (TACE)-induced shedding of EGFR ligands [transforming growth factor (TGF-{alpha})]. Active ligands may be released by cleavage of membrane-anchored proligand (exemplified by pro-TGF-{alpha}). Binding of EGFR ligands in the extracellular domain in airway epithelial cells is followed by phosphorylation of tyrosine residues in the intracellular domain. Activated EGFR triggers a downstream cascade leading to mucin gene and protein synthesis. Rosiglitazone inhibits TNF-{alpha} and forms a heterodimer with retinoid X receptor (RXR) and peroxisome proliferator-activated receptor (PPAR)-{gamma} receptor. This heterodimer binds PPAR response elements in genomic sequence upstream of PTEN, and PTEN expression is upregulated. The activation of PTEN results in a decrease of phosphatidylinositol 3,4,5-triphosphate (PIP3) concentration, which in turn leads to Akt pathway inactivation. TNFR, tumor necrosis factor receptor; PIP2, phosphatidylinositol 4, 5-biphosphate.

 

   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by Korea Research Foundation Grant KRF-2004-041-E00145.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. J. Shim, Div. of Pulmonary and Critical Care, Dept. of Internal Medicine, Korea Univ. Guro Hospital, #80, Guro-dong, Guro-gu, Seoul, Republic of Korea 135-702 (e-mail: jaejshim{at}kumc.or.kr)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Adler KB, Fischer BM, Wright DT, Cohn LA, and Becker S. Interactions between respiratory epithelial cells and cytokines: relationships to lung inflammation. Ann NY Acad Sci 725: 128–145, 1994.[ISI][Medline]
  2. Aljada A, Garg R, Ghanim H, Mohanty P, Hamouda W, Assian E, and Dandona P. Nuclear factor-kappaB suppressive and inhibitor-kappaB stimulatory effects of troglitazone in obese patients with type 2 diabetes: evidence of an antiinflammatory action? J Clin Endocrinol Metab 86: 3250–3256, 2001.[Abstract/Free Full Text]
  3. Atherton HC, Jones G, and Danahay H. IL-13-induced changes in the goblet cell density of human bronchial epithelial cell cultures: MAP kinase and phosphatidylinositol 3-kinase regulation. Am J Physiol Lung Cell Mol Physiol 285: L730–L739, 2003.[Abstract/Free Full Text]
  4. Backman S, Stambolic V, and Mak T. PTEN function in mammalian cell size regulation. Curr Opin Neurobiol 12: 516–522, 2002.[CrossRef][ISI][Medline]
  5. Baeuerle PA and Baltimore D. NF-kappa B: ten years after. Cell 87: 13–20, 1996.[CrossRef][ISI][Medline]
  6. Burgel PR and Nadel JA. Roles of epidermal growth factor receptor activation in epithelial cell repair and mucin production in airway epithelium. Thorax 59: 992–996, 2004.[Abstract/Free Full Text]
  7. Cantley LC. The phosphoinositide 3-kinase pathway. Science 296: 1655–1657, 2002.[Abstract/Free Full Text]
  8. Cantrell DA. Phosphoinositide 3-kinase signalling pathways. J Cell Sci 114: 1439–1445, 2001.[Abstract]
  9. Chan TO, Rittenhouse SE, and Tsichlis PN. AKT/PKB and other D3 phosphoinositide-regulated kinases: kinase activation by phosphoinositide-dependent phosphorylation. Annu Rev Biochem 68: 965–1014, 1999.[CrossRef][ISI][Medline]
  10. Chen G and Goeddel DV. TNF-R1 signaling: a beautiful pathway. Science 296: 1634–1635, 2002.[Abstract/Free Full Text]
  11. Chen R, Lim JH, Jono H, Gu XX, Kim YS, Basbaum CB, Murphy TF, and Li JD. Nontypeable Haemophilus influenzae lipoprotein P6 induces MUC5AC mucin transcription via TLR2-TAK1-dependent p38 MAPK-AP1 and IKKbeta-IkappaBalpha-NF-kappaB signaling pathways. Biochem Biophys Res Commun 324: 1087–1094, 2004.[CrossRef][ISI][Medline]
  12. Chung SW, Kang BY, Kim SH, Pak YK, Cho D, Trinchieri G, and Kim TS. Oxidized low density lipoprotein inhibits interleukin-12 production in lipopolysaccharide-activated mouse macrophages via direct interactions between peroxisome proliferator-activated receptor-gamma and nuclear factor-kappa B. J Biol Chem 275: 32681–32687, 2000.[Abstract/Free Full Text]
  13. Cunard R, Ricote M, DiCampli D, Archer DC, Kahn DA, Glass CK, and Kelly CJ. Regulation of cytokine expression by ligands of peroxisome proliferator activated receptors. J Immunol 168: 2795–2802, 2002.[Abstract/Free Full Text]
  14. Davies MA, Kim SJ, Parikh NU, Dong Z, Bucana CD, and Gallick GE. Adenoviral-mediated expression of MMAC/PTEN inhibits proliferation and metastasis of human prostate cancer cells. Clin Cancer Res 8: 1904–1914, 2002.[Abstract/Free Full Text]
  15. Deshmukh HS, Case LM, Wesselkamper SC, Borchers MT, Martin L, Shertzer HG, Nadel JA, and Leikauf GD. Metalloproteinases mediate mucin 5AC expression by epidermal growth factor receptor activation. Am J Respir Crit Care Med 171: 305–314, 2005.[Abstract/Free Full Text]
  16. Desvergne B and Wahli W. Peroxisome proliferator-activated receptors: nuclear control of metabolism. Endocr Rev 20: 649–688, 1999.[Abstract/Free Full Text]
  17. Forman BM, Tontonoz P, Chen J, Brun RP, Spiegelman BM, and Evans RM. 15-Deoxy-delta 12, 14-prostaglandin J2 is a ligand for the adipocyte determination factor PPAR gamma. Cell 83: 803–812, 1995.[CrossRef][ISI][Medline]
  18. Fruman DA and Cantley LC. Phosphoinositide 3-kinase in immunological systems. Semin Immunol 14: 7–18, 2002.[CrossRef][ISI][Medline]
  19. Goetze S, Eilers F, Bungenstock A, Kintscher U, Stawowy P, Blaschke F, Graf K, Law RE, Fleck E, and Grafe M. PPAR activators inhibit endothelial cell migration by targeting Akt. Biochem Biophys Res Commun 293: 1431–1437, 2002.[CrossRef][ISI][Medline]
  20. Goldkorn T, Balaban N, Matsukuma K, Chea V, Gould R, Last J, Chan C, and Chavez C. EGF-receptor phosphorylation and signaling are targeted by H2O2 redox stress. Am J Respir Cell Mol Biol 19: 786–798, 1998.[Abstract/Free Full Text]
  21. Gu J, Tamura M, and Yamada KM. Tumor suppressor PTEN inhibits integrin- and growth factor-mediated mitogen-activated protein (MAP) kinase signaling pathways. J Cell Biol 143: 1375–1383, 1998.[Abstract/Free Full Text]
  22. Hlobilkova A, Knillova J, Bartek J, Lukas J, and Kolar Z. The mechanism of action of the tumour suppressor gene PTEN. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 147: 19–25, 2003.[Medline]
  23. Hong G, Davis B, Khatoon N, Baker SF, and Brown J. PPAR gamma-dependent anti-inflammatory action of rosiglitazone in human monocytes: suppression of TNF alpha secretion is not mediated by PTEN regulation. Biochem Biophys Res Commun 303: 782–787, 2003.[CrossRef][ISI][Medline]
  24. Jones R, Bolduc P, and Reid L. Goblet cell glycoprotein and tracheal gland hypertrophy in rat airways: the effect of tobacco smoke with or without the anti-inflammatory agent phenylmethyloxadiazole. Br J Exp Pathol 54: 229–239, 1973.[ISI][Medline]
  25. Kane LP, Shapiro VS, Stokoe D, and Weiss A. Induction of NF-kappaB by the Akt/PKB kinase. Curr Biol 9: 601–604, 1999.[CrossRef][ISI][Medline]
  26. Katso R, Okkenhaug K, Ahmadi K, White S, Timms J, and Waterfield MD. Cellular function of phosphoinositide 3-kinases: implications for development, homeostasis, and cancer. Annu Rev Cell Dev Biol 17: 615–675, 2001.[CrossRef][ISI][Medline]
  27. Kliewer SA, Umesono K, Noonan DJ, Heyman RA, and Evans RM. Convergence of 9-cis retinoic acid and peroxisome proliferator signalling pathways through heterodimer formation of their receptors. Nature 358: 771–774, 1992.[CrossRef][Medline]
  28. Klingler-Hoffmann M, Bukczynska P, and Tiganis T. Inhibition of phosphatidylinositol 3-kinase signaling negates the growth advantage imparted by a mutant epidermal growth factor receptor on human glioblastoma cells. Int J Cancer 105: 331–339, 2003.[CrossRef][ISI][Medline]
  29. Kohri K, Ueki IF, and Nadel JA. Neutrophil elastase induces mucin production by ligand-dependent epidermal growth factor receptor activation. Am J Physiol Lung Cell Mol Physiol 283: L531–L540, 2002.[Abstract/Free Full Text]
  30. Lewis JD, Lichtenstein GR, Stein RB, Deren JJ, Judge TA, Fogt F, Furth EE, Demissie EJ, Hurd LB, Su CG, Keilbaugh SA, Lazar MA, and Wu GD. An open-label trial of the PPAR-gamma ligand rosiglitazone for active ulcerative colitis. Am J Gastroenterol 96: 3323–3328, 2001.[ISI][Medline]
  31. Lora JM, Zhang DM, Liao SM, Burwell T, King AM, Barker PA, Singh L, Keaveney M, Morgenstern J, Gutierrez-Ramos JC, Coyle AJ, and Fraser CC. Tumor necrosis factor-alpha triggers mucus production in airway epithelium through an IkappaB kinase beta-dependent mechanism. J Biol Chem 280: 36510–36517, 2005.[Abstract/Free Full Text]
  32. Myers MP, Stolarov JP, Eng C, Li J, Wang SI, Wigler MH, Parsons R, and Tonks NK. P-TEN, the tumor suppressor from human chromosome 10q23, is a dual-specificity phosphatase. Proc Natl Acad Sci USA 94: 9052–9057, 1997.[Abstract/Free Full Text]
  33. Nadel JA. Role of epidermal growth factor receptor activation in regulating mucin synthesis. Respir Res 2: 85–89, 2001.[CrossRef][ISI][Medline]
  34. Nakajima A, Wada K, Miki H, Kubota N, Nakajima N, Terauchi Y, Ohnishi S, Saubermann LJ, Kadowaki T, Blumberg RS, Nagai R, and Matsuhashi N. Endogenous PPAR gamma mediates anti-inflammatory activity in murine ischemia-reperfusion injury. Gastroenterology 120: 460–469, 2001.[CrossRef][ISI][Medline]
  35. O'Donnell RA, Richter A, Ward J, Angco G, Mehta A, Rousseau K, Swallow DM, Holgate ST, Djukanovic R, Davies DE, and Wilson SJ. Expression of ErbB receptors and mucins in the airways of long term current smokers. Thorax 59: 1032–1040, 2004.[Abstract/Free Full Text]
  36. O'Mahony A, Lin X, Geleziunas R, and Greene WC. Activation of the heterodimeric IkappaB kinase alpha (IKKalpha)-IKKbeta complex is directional: IKKalpha regulates IKKbeta under both basal and stimulated conditions. Mol Cell Biol 20: 1170–1178, 2000.[Abstract/Free Full Text]
  37. Patel L, Pass I, Coxon P, Downes CP, Smith SA, and Macphee CH. Tumor suppressor and anti-inflammatory actions of PPARgamma agonists are mediated via upregulation of PTEN. Curr Biol 11: 764–768, 2001.[CrossRef][ISI][Medline]
  38. Ricote M, Li AC, Willson TM, Kelly CJ, and Glass CK. The peroxisome proliferator-activated receptor-gamma is a negative regulator of macrophage activation. Nature 391: 79–82, 1998.[CrossRef][Medline]
  39. Rieusset J, Roques M, Bouzakri K, Chevillotte E, and Vidal H. Regulation of p85alpha phosphatidylinositol-3-kinase expression by peroxisome proliferator-activated receptors (PPARs) in human muscle cells. FEBS Lett 502: 98–102, 2001.[CrossRef][ISI][Medline]
  40. Rogers DF. Mucus hypersecretion in chronic obstructive pulmonary disease. Novartis Found Symp 234: 65–77, 2001.[ISI][Medline]
  41. Shao MX, Nakanaga T, and Nadel JA. Cigarette smoke induces MUC5AC mucin overproduction via tumor necrosis factor-{alpha}-converting enzyme in human airway epithelial (NCI-H292) cells. Am J Physiol Lung Cell Mol Physiol 287: L420–L427, 2004.[Abstract/Free Full Text]
  42. Shim JJ, Dabbagh K, Ueki IF, Dao-Pick T, Burgel PR, Takeyama K, Tam DC, and Nadel JA. IL-13 induces mucin production by stimulating epidermal growth factor receptors and by activating neutrophils. Am J Physiol Lung Cell Mol Physiol 280: L134–L140, 2001.[Abstract/Free Full Text]
  43. Stambolic V, Suzuki A, de la Pompa JL, Brothers GM, Mirtsos C, Sasaki T, Ruland J, Penninger JM, Siderovski DP, and Mak TW. Negative regulation of PKB/Akt-dependent cell survival by the tumor suppressor PTEN. Cell 95: 29–39, 1998.[CrossRef][ISI][Medline]
  44. Takeyama K, Dabbagh K, Jeong Shim J, Dao-Pick T, Ueki IF, and Nadel JA. Oxidative stress causes mucin synthesis via transactivation of epidermal growth factor receptor: role of neutrophils. J Immunol 164: 1546–1552, 2000.[Abstract/Free Full Text]
  45. Takeyama K, Dabbagh K, Lee HM, Agusti C, Lausier JA, Ueki IF, Grattan KM, and Nadel JA. Epidermal growth factor system regulates mucin production in airways. Proc Natl Acad Sci USA 96: 3081–3086, 1999.[Abstract/Free Full Text]
  46. Takeyama K, Jung B, Shim JJ, Burgel PR, Dao-Pick T, Ueki IF, Protin U, Kroschel P, and Nadel JA. Activation of epidermal growth factor receptors is responsible for mucin synthesis induced by cigarette smoke. Am J Physiol Lung Cell Mol Physiol 280: L165–L172, 2001.[Abstract/Free Full Text]
  47. Von Knethen AA and Brune B. Delayed activation of PPARgamma by LPS and IFN-gamma attenuates the oxidative burst in macrophages. FASEB J 15: 535–544, 2001.[Abstract/Free Full Text]
  48. West KA, Linnoila IR, Belinsky SA, Harris CC, and Dennis PA. Tobacco carcinogen-induced cellular transformation increases activation of the phosphatidylinositol 3'-kinase/Akt pathway in vitro and in vivo. Cancer Res 64: 446–451, 2004.[Abstract/Free Full Text]
  49. Willson TM, Cobb JE, Cowan DJ, Wiethe RW, Correa ID, Prakash SR, Beck KD, Moore LB, Kliewer SA, and Lehmann JM. The structure-activity relationship between peroxisome proliferator-activated receptor gamma agonism and the antihyperglycemic activity of thiazolidinediones. J Med Chem 39: 665–668, 1996.[CrossRef][ISI][Medline]
  50. Xia X and Serrero G. Inhibition of adipose differentiation by phosphatidylinositol 3-kinase inhibitors. J Cell Physiol 178: 9–16, 1999.[CrossRef][ISI][Medline]
  51. Yu CF, Liu ZX, and Cantley LG. ERK negatively regulates the epidermal growth factor-mediated interaction of Gab1 and the phosphatidylinositol 3-kinase. J Biol Chem 277: 19382–19388, 2002.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 ChestHome page
M. G. Belvisi and D. J. Hele
Peroxisome Proliferator-Activated Receptors as Novel Targets in Lung Disease
Chest, July 1, 2008; 134(1): 152 - 157.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. Rev.Home page
T. Yoshida and R. M. Tuder
Pathobiology of Cigarette Smoke-Induced Chronic Obstructive Pulmonary Disease
Physiol Rev, July 1, 2007; 87(3): 1047 - 1082.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
291/1/L84    most recent
00388.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (7)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lee, S. Y.
Right arrow Articles by Shim, J. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lee, S. Y.
Right arrow Articles by Shim, J. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2006 by the American Physiological Society.