Advanced
15-Hydroxyeicosatetraenoic Acid Inhibits Phorbol-12-Myristate-13-Acetate-Induced MUC5AC Expression in NCI-H292 Respiratory Epithelial Cells
15-Hydroxyeicosatetraenoic Acid Inhibits Phorbol-12-Myristate-13-Acetate-Induced MUC5AC Expression in NCI-H292 Respiratory Epithelial Cells
Journal of Microbiology and Biotechnology. 2015. May, 25(5): 589-597
Copyright © 2015, The Korean Society For Microbiology And Biotechnology
  • Received : January 20, 2015
  • Accepted : February 02, 2015
  • Published : May 28, 2015
Download
PDF
e-PUB
PubReader
PPT
Export by style
Article
Author
Metrics
Cited by
TagCloud
About the Authors
Yong-Seok Song
Man Sub Kim
Dong Hun Lee
Doek-Kun Oh
Do-Young Yoon
ydy4218@konkuk.ac.kr

Abstract
It has been reported that overexpression of MUC5AC induced by excessive inflammation leads to airway obstruction in respiratory diseases such as chronic obstructive pulmonary disease and asthma. 15-Hydroxyeicosatetraenoic acid (15-HETE) has been reported to have anti-inflammatory effects, but the role of 15-HETE in respiratory inflammation has not been determined. Therefore, the aim of this study was to investigate the effects of 15-HETE on MUC5AC expression and related pathways. In this study, phorbol-12-myristate-13-acetate (PMA) was used to stimulate NCI-H292 bronchial epithelial cells in order to examine the effects of 15-HETE. 15-HETE inhibited PMA-induced expression of MUC5AC mRNA and secretion of MUC5AC protein. Moreover, 15-HETE regulated matrix metallopeptidase 9 (MMP-9), mitogen-activated protein kinase kinase (MEK), and extracellular signal-regulated kinase (ERK). In addition, 15-HETE decreased the nuclear translocation of specificity protein-1 (Sp-1) transcription factor and nuclear factor κB (NF-κB). Furthermore, 15-HETE enhanced the transcriptional activity of peroxisome proliferator-activated receptor gamma (PPAR γ ) as a PPAR γ agonist. This activity reduced the phosphorylation of protein kinase B (PΚB/Akt) by increasing the expression of phosphatase and tensin homolog (PTEN). In conclusion, 15-HETE regulated MUC5AC expression via modulating MMP-9, MEK/ERK/Sp-1, and PPAR γ /PTEN/Akt signaling pathways in PMA-treated respiratory epithelial cells.
Keywords
Introduction
Thickening of the airway mucus layer caused by chronic inflammation is an important feature of respiratory diseases such as asthma and chronic obstructive pulmonary disease [9 , 18] . In these diseases, overproduction of mucus causes a narrowed airway and impaired lung function [1] . Mucins are glycoproteins that are major components of mucus. Among the 20 genes in the mucin subfamily, the MUC5AC and MUC5B genes encode the major glycoproteins of the mucus layer in normal airways [15] . However, MUC5AC overexpression was reported to be a major feature in asthma [34] .
15-Hydroxyeicosatetraenoic acid (15-HETE) is a major metabolite of arachidonic acid formed by 15-lipoxygenase (15-LOX) [4] . 15-HETE has been investigated for its anti cancer effect [30] , and was also reported to have antiinflammatory effects [41] . Although 15-lipoxygenase-1 (15-LOX-1) is reported to be implicated in asthma [45] , the effect of 15-HETE on respiratory inflammation has not been elucidated.
In order to examine the effect of 15-HETE on MUC5AC, we investigated the level of MUC5AC expression and related pathways in NCI-H292 bronchial epithelial cells, which express MUC5AC by stimulus, such as phorbol-12-myristate-13-acetate (PMA) [14] . Because of its effectiveness, PMA has been used to stimulate MUC5AC expression in a number of studies [10 , 14 , 27] . Thus, our study was focused on verifying the regulatory effects of 15-HETE on PMA-induced MUC5AC expression in NCI-H292 cells. These results may have implications in pharmacological research where some anti-inflammatory drugs have been reported to stimulate 15-HETE production [20] .
Materials and Methods
- Cell Culture and Stimulation
NCI-H292 human mucoepidermoid pulmonary carcinoma cells were obtained from ATCC (American Type Culture Collection, Manassas, VA, USA) and cultured in RPMI-1640 (Hyclone, Logan, UT, USA) supplemented with 10% (v/v) fetal calf serum (FBS) and penicillin (100 U/ml) and streptomycin (100 µg/ml) (Hyclone). These cells were cultured in 6-well plates until confluent and then serum-starved for 24 h. After serum starvation, the NCI-H292 cells were treated with 15-HETE (produced and donated by Dr. Oh DK, Konkuk University, Seoul, Korea as previously reported [16] ) for 1 h, and then stimulated with PMA (10 nM; Sigma, St Louis, MO, USA) for 24 h.
- Detection of MUC5AC Protein
NCI-H292 cells were cultured in 6-well plates. After stimulation (as described above), cultured media were collected and MUC5AC protein was analyzed by dot-blot assay as previously described [19] . Briefly, 3 µl of supernatant was loaded onto a nitrocellulose membrane. After 1 h of blocking with 5% skim milk, the membrane was incubated with anti-MUC5AC antibodies (clone 45M1; Neomarkers, Fremont, CA, USA) for 1 h at room temperature. The membrane was then incubated with horseradish peroxidaseconjugated goat anti-mouse antibodies for 30 min at room temperature. Chemiluminescence images were taken by using an ATTO cooled CCD camera system Ez-Capture MG (Atto Co., Tokyo, Japan) and the intensity of chemiluminescence was quantified with CS Analyzer (Atto Co.).
- RNA Isolation and Reverse Transcription Polymerase Chain Reaction (RT-PCR)
Total RNAs were isolated with an R&A BLUE Total RNA Extraction kit (iNtRON, Seoul, Korea). cDNA was synthesized with oligo (dT) using M-MuLV reverse transcriptase (New England Biolabs, Ipswich, MA, USA). The sequences of primers were as follows: MUC5AC (5’-TGATCATCCAGCAGCAGGGCT-3’ and 5’-CCGAGCTCAGAGGACATATGGG-3’), matrix metallopeptidase-9 (MMP-9; 5’-CGCAGACATCGTCATCCAGT-3’ and 5’-GGATTGGCCTTGGAAGATGA-3’), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 5’-AGGTCGGAGTCAACGGATTT-3’ and 5’-ATGATGTTCTGGAGAGCCCC-3’). GAPDH was used as the RNA loading control. RT-PCR products were electrophoresed on 1% agarose gels and detected by ethidium bromide staining.
- Gelatin Zymography
The activity of MMP-9 in NCI-H292 cells was investigated by conducting gelatin zymography as described previously [24] . Briefly, 15 µl of supernatant was loaded into a 0.1% gelatin gel to determine the presence of MMP-9 (92 kDa). Electrophoresis was performed under non-denaturing conditions and at a constant voltage of 100 V for 90 min. The gel was washed with 2.5% Triton X-100 for 20 min, followed by two additional washes with distilled water for 10 min each. Then the gel was activated with a reaction buffer (50 mM Tris-HCl (pH 7.5), 10 mM CaCl 2 , 150 mM NaCl, 2 µM ZnCl 2 , 1% Triton X-100, and 0.002% NaN 3 ) at 37℃ for 24 h. The gels were stained with Coomassie Brilliant Blue R-250 (Sigma) and destained. Areas of gelatinolytic degradation by MMP-9 appeared as transparent bands. Images were taken by using an ATTO cooled CCD camera system EZ-Capture MG (Atto Co.) and the intensity of band was quantified by CS Analyzer (Atto Co.).
- Immunoblot Analysis
After treatment with PMA for 1 h, NCI-H292 cells were lysed with radio immunoprecipitation assay buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 1% NP-40, 0.1% sodium dodecyl sulfate, 0.25% sodium deoxycholate, 1 mM ethylenediaminetetraacetic acid, 1 mM ethylene glycol tetraacetic acid, 1 mM orthovanadate, aprotinin (10 µg/ml), and 0.4 mM phenylmethylsulfonyl fluoride). Equal amounts (50 µg) of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. Specific proteins were detected with respective primary antibodies to phosphorylated mitogen-activated protein kinase kinase (p-MEK), total MEK (T-MEK) (Cell Signaling, Danvers, MA, USA), phosphorylated extracellular signal-regulated kinase (p-ERK), total ERK (T-ERK), phosphorylated protein kinase B (p-PKB/Akt), total Akt (T-Akt), phosphatase and tensin homolog (PTEN), and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Images were taken by using an ATTO cooled CCD camera system EZ-Capture MG (Atto Co.) and the intensity of band was quantified by CS Analyzer (Atto Co.). The phosphorylation levels of MEK, ERK, and Akt were analyzed by measuring the ratio of respective phosporylated level to total level of each protein. The level of PTEN expression was quantified and normalized to GAPDH.
- Nuclear and Cytoplasmic Fractionation
After 1 h treatment with PMA, NCI-H292 cells were harvested and fractionated with NE-PER Nuclear and Cytoplasmic Extraction Reagent (Thermo Fisher Scientific, Inc., Rockford, IL, USA) according to the manufacturer’s instructions. Lysate proteins were analyzed by immunoblot analysis. Primary antibodies to specificity protein-1 (Sp-1), nuclear factor κB p65 (NF-κB p65), poly (ADP-ribose) polymerase (PARP), and GAPDH were used to determine the translocation of transcription factors. PARP and GAPDH were used as markers for the nuclear and cytoplasmic fractions, respectively.
- Immunofluorescence
NCI-H292 cells were seeded onto sterile cover slides and cultured. After pre-incubation with 15-HETE (1 µM) and stimulation with PMA (10 nM) for 1 h, the cells were fixed at room temperature for 10 min with 100% acetone. After washing with phosphatebuffered saline (PBS), the slides were blocked with 1% bovine serum albumin in PBS for 1 h at room temperature. After washing, the cells were incubated with Sp-1 antibodies diluted at 1:100 in PBS containing 0.1% bovine serum albumin for overnight at 4℃ normal rabbit IgG antibodies were used to determine non-specific binding. After washing three times with PBS, the cells were incubated with fluorescein isothiocyanate-labeled rabbit secondary antibodies diluted 1:200 folds for 1 h at room temperature. Then, cells were stained with 4,6-diamidino-2-phenylindole for 10 sec at room temperature. The cover slides containing the cells were mounted with a mounting agent (Merck Millipore, Darmstadt, Germany). Immunofluorescence was examined using a fluorescence microscope (BX61-32FDIC; Olympus, Tokyo, Japan).
- Transcriptional Activity Assay
In order to examine the effect of 15-HETE on the transcriptional activity of PPARγ, human embryonic kidney (HEK) 293 cells were used to conduct the luciferase assay as previously described [6 , 17] . In brief, the cells were cultured in Dulbecco’s modified Eagle’s medium (Hyclone) containing 10% FBS and antibiotics. Cells were seeded in 24-well plates (1.0 × 10 5 cells per well). The next day, cells were co-transfected with pcDNA3.1 vector plasmid expressing PPARγ, PPAR response element (PPRE) × 3-thymidine kinase-luciferase reporter constructs, and pRL Renilla Luciferase Reporter Vector (Promega, Madison, WI, USA). After 24 h, the cells were treated with 15-HETE (1 µM) in the absence or presence of troglitazone (3 µM). After another 24 h, the cells were harvested and assayed with a Dual-Luciferase Reporter Gene Assay Kit (Promega). The assay results were described in relative luciferase activity units, obtained by the ratio of the expression of firefly luciferase to Renilla luciferase.
- Statistical Analysis
Data were analyzed by using Student’s t -test to evaluate significant differences between experimental and control groups. A p -value of <0.05 was considered statistically significant. The data were presented as the mean ± SD of at three independent experiments.
Results
- Effects of 15-HETE on PMA-Induced MUC5AC mRNA and Protein Levels
To examine whether 15-HETE would have an inhibitory effect on MUC5AC production, NCI-H292 cells were treated with 15-HETE for 1 h and stimulated with PMA (10 nM) for 24 h. The MUC5AC mRNA level was determined by RT-PCR, and the secreted MUC5AC level in culture media was analyzed by a dot-blot assay. PMA enhanced the MUC5AC mRNA expression ( Fig. 1 A) and secreted protein levels ( Figs. 1 B and 1 C). However, 15-HETE reduced the PMA-induced MUC5AC mRNA and protein levels ( Fig. 1 ). Therefore, these results revealed that 15-HETE inhibited PMA-induced MUC5AC expression.
PPT Slide
Lager Image
Effect of 15-HETE on PMA-induced MUC5AC expression. NCI-H292 cells were pre-treated with 15-HETE (0, 0.01, 0.1, 0.5, and 1 µM) for 1 h and stimulated with PMA (10 nM) for 24 h. (A) After stimulation, MUC5AC and GAPDH mRNA expression was analyzed by RT-PCR. GAPDH was used as an internal control. Representative bands are shown from more than three independent experiments. (B) MUC5AC protein in cell supernatants was measured by dot-blot analysis with anti-MUC5AC antibodies. A representative blot from one of four independent experiments is shown. (C) Graphs of four dot-blot analyses (mean ± SD). Band densities were normalized to the group without treatment of 15-HETE and PMA and presented as a fold change. *p < 0.05; **p < 0.01 vs. cells treated with only PMA.
- Effect of 15-HETE on PMA-Induced MMP-9 Expression
PMA has been reported to activate MMP-9 [37] , which cleaves transforming growth factor alpha (TGF-α) into the active form, the substrate of epidermal growth factor receptor (EGFR) [12] that has a role in MUC5AC production [5] . To investigate the effect of 15-HETE on MMP-9 mRNA expression, RT-PCR was conducted and the MMP-9 protein level was analyzed by gelatin zymography. The PMA-induced activation of NCI-H292 cells resulted in the up-regulation of MMP-9 mRNA expression ( Fig. 2 A) and protein level ( Figs. 2 B and 2 C). However, the induced MMP-9 levels were decreased by 15-HETE ( Fig 2 ). Taken together, these data showed that 15-HETE suppressed the PMA-induced MUC5AC expression.
PPT Slide
Lager Image
Effect of 15-HETE on PMA-activated MMP-9. NCI-H292 cells were pre-incubated with 15-HETE (0, 0.01, 0.1, 0.5, and 1 µM) for 1 h and treated with PMA (10 nM) for 24 h. After stimulation, (A) MMP-9 and GAPDH mRNA were analyzed by RT-PCR using specific primers. GAPDH was used as the internal control. Representative bands are shown from more than three independent experiments. (B) The levels of MMP-9 in cell culture media were examined by gelatin zymography. A representative band is shown from three independent experiments. (C) Graphs of three zymography analyses (mean ± SD). Band densities were normalized to the group without treatment of 15-HETE and PMA and presented as a fold change. *p < 0.05; **p < 0.01 vs. cells treated with only PMA.
- Effect of 15-HETE on PMA-Activated MEK/ERK Signaling Pathway
The MEK/ERK signaling pathway has been reported to mediate EGFR signaling to MUC5AC expression [33] . In addition, PMA has been reported to induce MUC5AC via a phosphorylation cascade of the MEK/ERK pathway [14] . To examine the effect of 15-HETE on the PMA-induced MEK/ERK pathway, immunoblot analysis was conducted to assess the phosphorylation level of MEK and ERK. As shown in Fig. 3 , the downregulation of MEK and ERK phosphorylation that was increased by PMA was observed in cells treated with 15-HETE compared with control cells stimulated with only PMA. These results revealed that 15-HETE inhibited the MEK/ERK signaling pathway in PMA-stimulated cells.
PPT Slide
Lager Image
Effect of 15-HETE on PMA-induced MEK/ERK pathway. (A) NCI-H292 cells were pre-treated with 15-HETE (0, 0.01, 0.1, 0.5, and 1 µM) for 1 h and stimulated with PMA (10 nM) for 1 h. The cells were harvested, and phosphorylated MEK (p-MEK), total MEK (T-MEK), phosphorylated ERK (p-ERK), and total ERK (T-ERK) were detected by immunoblot analysis. Representative bands are shown from more than three independent experiments. Graphs summarizing the densitometric results of three independent experiments are presented as the mean ± SD of the ratio of p-MEK to T-MEK (B) and the ratio of p-ERK to T-ERK (C).
- Effects of 15-HETE on PMA-Induced Translocation of Transcription Factors
Sp-1 transcription factor has been reported to be activated by PMA via the MEK/ERK signaling cascade and translocated into the nucleus, inducing MUC5AC expression [14] . In order to determine the effect of 15-HETE on the translocation of Sp-1, 15-HETE-and PMA-treated cells were fractionated into the cytoplasm and nuclear fractions, and each fraction was examined by immunoblot assay. As shown in the Fig. 4 A, PMA treatment activated the translocation of Sp-1. However, the increased Sp-1 level in the nuclear fraction was decreased in 15-HETE-treated cells. In addition, immunofluorescence assays were performed to visualize the effect of 15-HETE on the translocation of Sp-1. As shown in Fig. 4 B, the PMA-induced nuclear localization of Sp-1 was reduced by 15-HETE. Furthermore, 15-HETE exerted an inhibitory effect on translocation of NF-κB p65 ( Fig. 4 A) which has been reported to be activated and translocated into the nuclear by PMA, resulting in MUC5AC expression [26] . Taken together, these data showed that 15-HETE inhibited the translocation of Sp-1 and NF-κB into the nucleus.
PPT Slide
Lager Image
Effects of 15-HETE on PMA-induced translocation of Sp-1 and NF-κB into the nucleus. (A) NCI-H292 cells were pre-treated with 15-HETE (1 µM) for 1 h and stimulated with PMA (10 nM) for 1 h. After treatment, cells were harvested and fractionated into nuclear and cytosolic fractions and analyzed by immunoblot analysis to detect the level of Sp-1, NF-κB (p65), PARP, and GAPDH in each fraction. PARP and GAPDH were used as markers for the nuclear and cytosolic fractions, respectively. Representative bands are shown from three independent experiments. (B) NCI-H292 cells were seeded on cover slides and cultured. The cells were pre-incubated with 15-HETE (1 µM) and stimulated with PMA (10 nM) for 1 h. After treatment, the cells were fixed with 100% acetone for 10 min and analyzed by immunofluorescence to detect the translocation of Sp-1 transcription factors. Representative images are presented from three independent experiments.
- Effects of 15-HETE on PTEN and p-Akt Expression as a PPARγ Agonist
It has been reported that 15-HETE can act as a PPARγ agonist [35] . In addition, PPARγ agonists have been reported to inhibit mucin expression both in vitro and in vivo [36] and possible mechanisms have been suggested in that up-regulation of PTEN expression by PPARγ agonist decreases the Akt signaling pathway [25] , which has been reported to be involved in MUC5AC production [44] . Moreover, the phosphorylation of Akt is induced by several stimuli, such as epidermal growth factor (EGF) [43] and PMA [31] to activate Akt, resulting in the nuclear translocation of NFκB [26] .
Therefore, the effect of 15-HETE on PPARγ as an agonist was examined by conducting a luciferase reporter assay using a PPRE-luciferase reporter construct. The effect of 15-HETE on Akt phosphorylation was also examined by immunoblot analysis. 15-HETE increased the activity of PPARγ, and the activity was more increased when 15-HETE was co-administered with troglitazone, a wellknown PPARγ agonist [11] ( Fig. 5 A). As shown in Fig. 5 B, 15-HETE increased the expression of PTEN and suppressed Akt phosphorylation in PMA-treated cells. In addition, the NF-κB translocation, which is related to the Akt pathway, was decreased by 15-HETE ( Fig. 4 A). These data indicate that 15-HETE affected the Akt pathway by increasing PTEN expression as a PPARγ agonist.
PPT Slide
Lager Image
Effect of 15-HETE on the PTEN/Akt pathway as a PPARγ agonist. (A) HEK-293 cells were seeded in a 24-well plate (1.0 × 105 cells per well). After 24 h of incubation, the cells were transfected with vector plasmids expressing PPARγ, PPAR response element (PPRE) × 3-thymidine kinase-luciferase reporter constructs, and Renilla luciferase control vector pRL. After another 24 h, cells were treated with 15-HETE (1 µM) and troglitazone (3 µM) for 24 h. The cells were harvested and the transcriptional activity of PPARγ was determined by a luciferase assay. Means ± SD of three independent experiments are shown. *p < 0.05. (B) NCI-H292 cells were pre-incubated with 15-HETE (0, 0.01, 0.1, 0.5, and 1 µM) for 1 h and stimulated with PMA (10 nM) for 1 h. After stimulation, cells were harvested and phosphorylated Akt (p-Akt), total Akt (T-Akt), PTEN, and GAPDH were detected by immunoblot analysis. GAPDH was used as the internal control. Representative bands are presented from more than three independent experiments. Graphs summarizing densitometric results of three independent experiments are presented as the mean ± SD of the ratio of p-Akt to T-Akt (C) and the ratio of PTEN to GAPDH (D).
Discussion
In this study, we demonstrated the effects of 15-HETE on MUC5AC expression. We investigated the PMA-induced signaling pathway in relation to MUC5AC expression in NCI-H292 cells. Our results indicated that 15-HETE has an anti-inflammatory effect by blocking the PMA-induced MEK/ERK/Sp-1 signaling pathway and enhancing PTEN expression.
Common chronic airway disorders such as chronic obstructive pulmonary disease and asthma are associated with the overproduction of mucin glycoproteins. MUC5AC, the major mucin in the respiratory epithelium, is stimulated by several factors such as IL-13 [46] , EGF [43] , and PMA [14] . The stimulation of MUC5AC by PMA is dependent on the activation of the EGFR, an important step in mucin expression in the respiratory epithelium [5] . In response to EGFR activation, the MEK/ERK signaling cascade is upregulated to induce translocation of the Sp-1 transcription factor into the nucleus [14] . In this study, we demonstrated that the PMA-induced MEK/ERK/Sp-1 signaling pathway was inhibited by 15-HETE.
15-HETE, an oxidized eicosanoid, is a major product of 15-LOXs. LOXs are a family of enzymes that catalyze the oxygenation of polyunsaturated fatty acids such as arachidonic acid. To date, two 15-LOXs have been reported; one is 15-LOX-1, and the other is 15-LOX-2, which exclusively produces 15-HETE [4 , 38] . 15-HETE has been investigated as a biological modulator in various pathophysiological conditions [13] . Although there are some studies suggesting the role of 15-HETE, the one possible explanation is that 15-HETE can mediate an anti-inflammatory effect [21 , 41] . Our results also imply that 15-HETE exerts anti-inflammatory effects via inhibiting MUC5AC expression in PMA-treated NCI-H292 cells.
It has been recently reported that inhibiting 15-LOX-1 expression decreases IL-13-induced MUC5AC expression, and phosphatidylethanolamine (PE)-conjugated 15-HETE activates MUC5AC production [46] . However, 15-HETE is mainly conjugated with phosphatidylinositol rather than PE in human tracheal epithelial cells [2] . Furthermore, 15-LOX-1 preferentially metabolizes linoleic acid to 13-hydroperoxyoctadecadienoic acid [22] . Moreover, 15-LOX-1 has been reported to be less efficient than 15-LOX-2 in the production of 15-HETE [23] . Therefore, 15-HETE has been reported to be synthesized mainly by 15-LOX-2 rather than 15-LOX-1 [28] and the role of 15-HETE in mucin expression remains to be determined. In addition, further study will be required for the identification of the role of 15-LOX-2 in airway inflammation.
Besides the anti-inflammatory effects, 15-HETE has been reported to bind to PPARγ [40] and activates PPARγ [3 , 35] . PPARγ is a subtype of the PPAR (α, β, and γ) family, ligand-inducible transcription factors. The activation of PPARs by certain ligands leads to heterodimerization with the retinoid X receptor, and the dimerized complex binds to a specific element, PPAR response element (PPRE), located in the promoter domain of the target genes [7 , 8] . PPARγ has been well known as a key regulator of adipogenesis, but later studies have shown that PPARγ has anti-inflammatory effects such as regulating inflammatory cytokines [29] .
In a recent study, the synthetic PPARγ agonist rosiglitazone decreased the MUC5AC expression induced by a solution of cigarette smoke in NCI-H292 cells [25] . This regulatory effect of rosiglitazone was mediated by increased PTEN expression, resulting in the reduction of Akt activation [25] . It has been reported that the Akt pathway activated by PI3K also contributes to the expression of MUC5AC [39] . Activated Akt induces the phosphorylation of IκB, resulting in upregulation of NF-κB translocation that promotes MUC5AC production [44] . To regulate the activity of PI3K, PTEN has been reported to antagonize the PI3K-mediated reaction [25] . Furthermore, PPARγ has been shown to promote PTEN expression, resulting in the down-regulation of PI3K activity [32] . Our study showed that 15-HETE also enhanced PPARγ activity and PTEN expression, which decreased Akt phosphorylation. Therefore, these findings suggest a regulatory effect of 15-HETE on MUC5AC expression as a PPARγ agonist.
This is the first study to show a modulating effect of 15-HETE on PMA-induced MUC5AC expression in NCI-H292 bronchial epithelial cells. Our results suggest that 15-HETE may play an inhibitory role in chronic airway inflammation and pharmacological treatment. Nonsteroidal anti-inflammatory drugs (NSAIDs) have been reported to increase the production of 15-HETE in polymorphonuclear leukocytes [42] . In addition, aspirin has been shown to induce the generation of 15-HETE in aspirin-sensitive patients suffering from severe asthma [20] .
In conclusion, we have shown that treatment of airway epithelial cells with 15-HETE reduced PMA-induced mucin production by suppressing the MEK/ERK/Sp-1 pathway. 15-HETE also increased PTEN expression and reduced Akt phosphorylation as a PPARγ agonist. Therefore, we suggest that the effects of 15-HETE and NSAIDs on airway obstruction in chronic respiratory diseases need to be further investigated.
Acknowledgements
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean Government (2013-A423-0061). DYY was partially supported by the Priority Research Centers Program (2012-0006686) of NRF.
References
Aikawa T , Shimura S , Sasaki H , Ebina M , Takishima T 1992 Marked goblet cell hyperplasia with mucus accumulation in the airways of patients who died of severe acute asthma attack. Chest 101 916 - 921    DOI : 10.1378/chest.101.4.916
Alpert SE , Walenga RW 1993 Human tracheal epithelial cells selectively incorporate 15-hydroxyeicosatetraenoic acid into phosphatidylinositol. Am. J. Respir. Cell Mol. Biol. 8 273 - 281    DOI : 10.1165/ajrcmb/8.3.273
Blanquart C , Barbier O , Fruchart JC , Staels B , Glineur C 2003 Peroxisome proliferator-activated receptors: regulation of transcriptional activities and roles in inflammation. J. Steroid Biochem. Mol. Biol. 85 267 - 273    DOI : 10.1016/S0960-0760(03)00214-0
Brash AR , Boeglin WE , Chang MS 1997 Discovery of asecond 15S-lipoxygenase in humans. Proc. Natl. Acad. Sci. USA 94 6148 - 6152    DOI : 10.1073/pnas.94.12.6148
Burgel PR , Nadel JA 2008 Epidermal growth factor receptor-mediated innate immune responses and their roles in airway diseases. Eur. Respir. J. 32 1068 - 1081    DOI : 10.1183/09031936.00172007
Cho MC , Lee DH , Kim EJ , Lee JY , Kang JW , Song JH 2011 Novel PPARgamma partial agonists with weak activity and no cytotoxicity; identified by a simple PPARgamma ligand screening system. Mol. Cell Biochem. 358 75 - 83    DOI : 10.1007/s11010-011-0923-1
Cho MC , Lee K , Paik SG , Yoon DY 2008 Peroxisome proliferators-activated receptor (PPAR) modulators and metabolic disorders. PPAR Res. 2008 679137 -    DOI : 10.1155/2008/679137
Delerive P , Fruchart JC , Staels B 2001 Peroxisome proliferator-activated receptors in inflammation control. J. Endocrinol. 169 453 - 459    DOI : 10.1677/joe.0.1690453
Fahy JV 2002 Goblet cell and mucin gene abnormalities in asthma. Chest 122 320S - 326S    DOI : 10.1378/chest.122.6_suppl.320S
Garcia-Verdugo I , BenMohamed F , Tattermusch S , Leduc D , Charpigny G , Chignard M 2012 A role for 12R-lipoxygenase in MUC5AC expression by respiratory epithelial cells. Eur. Respir. J. 40 714 - 723    DOI : 10.1183/09031936.00023111
Ghanim H , Garg R , Aljada A , Mohanty P , Kumbkarni Y , Assian E 2001 Suppression of nuclear factor-kappaB and stimulation of inhibitor kappaB by troglitazone: evidence for an anti-inflammatory effect and a potential antiatherosclerotic effect in the obese. J. Clin. Endocrinol. Metab. 86 1306 - 1312
Gorgoulis V , Aninos D , Mikou P , Kanavaros P , Karameris A , Joardanoglou J 1992 Expression of EGF, TGF-alpha and EGFR in squamous cell lung carcinomas. Anticancer Res. 12 1183 - 1187
Hammond VJ , O VB 2012 Esterified eicosanoids: generation, characterization and function. Biochim. Biophys. Acta 1818 2403 - 2412    DOI : 10.1016/j.bbamem.2011.12.013
Hewson CA , Edbrooke MR , Johnston SL 2004 PMA induces the MUC5AC respiratory mucin in human bronchial epithelial cells,viaPKC, EGF/TGF-alpha, Ras/Raf, MEK, ERK and Sp1-dependent mechanisms. J. Mol. Biol. 344 683 - 695    DOI : 10.1016/j.jmb.2004.09.059
Hovenberg HW , Davies JR , Herrmann A , Linden CJ , Carlstedt I 1996 MUC5AC, but not MUC2, is a prominent mucin in respiratory secretions. Glycoconj. J. 13 839 - 847    DOI : 10.1007/BF00702348
Kim BJ , Shin KC , Oh DK 2014 Enzymatic production of 15-hydroxyeicosatetraenoic acid from arachidonic acid by using soybean lipoxygenase. J. Microbiol. Biotechnol. 24 359 - 362    DOI : 10.4014/jmb.1310.10054
Kim EJ , Lee DH , Kim HJ , Lee SJ , Ban JO , Cho MC 2012 Thiacremonone, a sulfur compound isolated from garlic, attenuates lipid accumulation partially mediated via AMPK activation in 3T3-L1 adipocytes. J. Nutr. Biochem. 23 1552 - 1558    DOI : 10.1016/j.jnutbio.2011.10.008
Kim V , Kelemen SE , Abuel-Haija M , Gaughan JP , Sharafkaneh A , Evans CM 2008 Small airway mucous metaplasia and inflammation in chronic obstructive pulmonary disease. COPD 5 329 - 338    DOI : 10.1080/15412550802522445
Kim YJ , Cho HJ , Shin WC , Song HA , Yoon JH , Kim CH 2014 Hypoxia-mediated mechanism of MUC5AC production in human nasal epithelia and its implication in rhinosinusitis. PLoS One 9 e98136 -    DOI : 10.1371/journal.pone.0098136
Kowalski ML , Ptasinska A , Jedrzejczak M , Bienkiewicz B , Cieslak M , Grzegorczyk J 2005 Aspirin-triggered 15-HETE generation in peripheral blood leukocytes is a specific and sensitive aspirin-sensitive patients identification test (ASPITest). Allergy 60 1139 - 1145    DOI : 10.1111/j.1398-9995.2005.00836.x
Kronke G , Katzenbeisser J , Uderhardt S , Zaiss MM , Scholtysek C , Schabbauer G 2009 12/15-lipoxygenase counteracts inflammation and tissue damage in arthritis. J. Immunol. 183 3383 - 3389    DOI : 10.4049/jimmunol.0900327
Kuhn H 1996 Biosynthesis, metabolization and biological importance of the primary 15-lipoxygenase metabolites 15-hydro(pero)XY-5Z,8Z,11Z,13E-eicosatetraenoic acid and 13-hydro(pero)XY-9Z,11E-octadecadienoic acid. Prog. Lipid Res. 35 203 - 226    DOI : 10.1016/S0163-7827(96)00008-2
Kuhn H , Barnett J , Grunberger D , Baecker P , Chow J , Nguyen B 1993 Overexpression, purification and characterization of human recombinant 15-lipoxygenase. Biochim. Biophys. Acta 1169 80 - 89    DOI : 10.1016/0005-2760(93)90085-N
Leber TM , Balkwill FR 1997 Zymography: a single-step staining method for quantitation of proteolytic activity on substrate gels. Anal. Biochem. 249 24 - 28    DOI : 10.1006/abio.1997.2170
Lee SY , Kang EJ , Hur GY , Jung KH , Jung HC , Lee SY 2006 Peroxisome proliferator-activated receptor-gamma inhibits cigarette smoke solution-induced mucin production in human airway epithelial (NCI-H292) cells. Am. J. Physiol. Lung Cell Mol. Physiol. 291 L84 - L90    DOI : 10.1152/ajplung.00388.2005
Lee SY , Lee HJ , Sikder MA , Shin HD , Kim JH , Chang GT 2012 Resveratrol inhibits mucin gene expression, production and secretion from airway epithelial cells. Phytother. Res. 26 1082 - 1087    DOI : 10.1002/ptr.3701
Li Q , Lei RX , Zhou XD , Kolosov VP , Perelman JM 2012 Regulation of PMA-induced MUC5AC expression by heparin in human bronchial epithelial cells. Mol. Cell Biochem. 360 383 - 391    DOI : 10.1007/s11010-011-1078-9
Mabalirajan U , Agrawal A , Ghosh B 2012 15-Lipoxygenase eicosanoids are the putative ligands for vanilloid receptors and peroxisome proliferator-activated receptors (PPARs). Proc. Natl. Acad. Sci. USA author reply E2. 109 E1 -
Martin H 2009 Role of PPAR-gamma in inflammation. Prospects for therapeutic intervention by food components. Mutat. Res. 669 1 - 7    DOI : 10.1016/j.mrfmmm.2009.06.009
Moreno JJ 2009 New aspects of the role of hydroxyeicosatetraenoic acids in cell growth and cancer development. Biochem. Pharmacol. 77 1 - 10    DOI : 10.1016/j.bcp.2008.07.033
Park SK , Hwang YS , Park KK , Park HJ , Seo JY , Chung WY 2009 Kalopanaxsaponin A inhibits PMA-induced invasion by reducing matrix metalloproteinase-9 via PI3K/Akt- and PKCdelta-mediated signaling in MCF-7 human breast cancer cells. Carcinogenesis 30 1225 - 1233    DOI : 10.1093/carcin/bgp111
Patel L , Pass I , Coxon P , Downes CP , Smith SA , Macphee CH 2001 Tumor suppressor and anti-inflammatory actions of PPARgamma agonists are mediated via upregulation of PTEN. Curr. Biol. 11 764 - 768    DOI : 10.1016/S0960-9822(01)00225-1
Perrais M , Pigny P , Copin MC , Aubert JP , Van Seuningen I 2002 Induction of MUC2 and MUC5AC mucins by factors of the epidermal growth factor (EGF) family is mediated by EGF receptor/Ras/Raf/extracellular signal-regulated kinase cascade and Sp1. J. Biol. Chem. 277 32258 - 32267    DOI : 10.1074/jbc.M204862200
Rose MC , Voynow JA 2006 Respiratory tract mucin genes and mucin glycoproteins in health and disease. Physiol. Rev. 86 245 - 278    DOI : 10.1152/physrev.00010.2005
Shappell SB , Gupta RA , Manning S , Whitehead R , Boeglin WE , Schneider C 2001 15S-Hydroxyeicosatetraenoic acid activates peroxisome proliferator-activated receptor gamma and inhibits proliferation in PC3 prostate carcinoma cells. Cancer Res. 61 497 - 503
Shen Y , Chen L , Wang T , Wen F 2012 PPARgamma as a potential target to treat airway mucus hypersecretion in chronic airway inflammatory diseases. PPAR Res. 2012 256874 -    DOI : 10.1155/2012/256874
Shin Y , Yoon SH , Choe EY , Cho SH , Woo CH , Rho JY , Kim JH 2007 PMA-induced up-regulation of MMP-9 is regulated by a PKCalpha-NF-kappaB cascade in human lung epithelial cells. Exper. Mol. Med. 39 97 - 105    DOI : 10.1038/emm.2007.11
Sigal E , Craik CS , Highland E , Grunberger D , Costello LL , Dixon RA , Nadel JA 1988 Molecular cloning and primary structure of human 15-lipoxygenase. Biochem. Biophys. Res. Commun. 157 457 - 464    DOI : 10.1016/S0006-291X(88)80271-7
Song S , Byrd JC , Guha S , Liu KF , Koul D , Bresalier RS 2011 Induction of MUC5AC mucin by conjugated bile acids in the esophagus involves the phosphatidylinositol 3-kinase/protein kinase C/activator protein-1 pathway. Cancer 117 2386 - 2397    DOI : 10.1002/cncr.25796
Subbarayan V , Krieg P , Hsi LC , Kim J , Yang P , Sabichi AL 2006 15-lipoxygenase-2 gene regulation by its product 15-(S)-hydroxyeicosatetraenoic acid through a negative feedback mechanism that involves peroxisome proliferator-activated receptor gamma. Oncogene 25 6015 - 6025    DOI : 10.1038/sj.onc.1209617
van Dijk AP , McCafferty DM , Wilson JH , Zijlstra FJ 1993 15-Hydroxy-eicosatetraenoic acid has minor anti-inflammatory properties in colitis. Agents Actions 38 C120 - C121    DOI : 10.1007/BF01991157
Vanderhoek JY , Ekborg SL , Bailey JM 1984 Nonsteroidal anti-inflammatory drugs stimulate 15-lipoxygenase/leukotriene pathway in human polymorphonuclear leukocytes. J. Allergy Clin. Immunol. 74 412 - 417    DOI : 10.1016/0091-6749(84)90140-4
Yamadori T , Ishii Y , Homma S , Morishima Y , Kurishima K , Itoh K 2012 Molecular mechanisms for the regulation of Nrf2-mediated cell proliferation in non-small-cell lung cancers. Oncogene 31 4768 - 4777    DOI : 10.1038/onc.2011.628
Yang J , Li Q , Zhou XD , Kolosov VP , Perelman JM 2011 Naringenin attenuates mucous hypersecretion by modulating reactive oxygen species production and inhibiting NF-kappaB activity via EGFR-PI3K-Akt/ERK MAPKinase signaling in human airway epithelial cells. Mol. Cell Biochem. 351 29 - 40    DOI : 10.1007/s11010-010-0708-y
Zhao J , Maskrey B , Balzar S , Chibana K , Mustovich A , Hu H 2009 Interleukin-13-induced MUC5AC is regulated by 15-lipoxygenase 1 pathway in human bronchial epithelial cells. Am. J. Respir. Crit. Care Med. 179 782 - 790    DOI : 10.1164/rccm.200811-1744OC
Zhao J , O’Donnell VB , Balzar S , St Croix CM , Trudeau JB , Wenzel SE 2011 15-Lipoxygenase 1 interacts with phosphatidylethanolamine-binding protein to regulate MAPK signaling in human airway epithelial cells. Proc. Natl. Acad. Sci. USA 108 14246 - 14251    DOI : 10.1073/pnas.1018075108