# Krüppel-Like Factor 5 Mediates Proinflammatory Cytokine Expression in Lipopolysaccharide-Induced Acute Lung Injury through Upregulation of Nuclear Factor-κB Phosphorylation In Vitro and In Vivo

Authors: Hsiu-Lin Chen; Inn-Wen Chong; Yi-Chen Lee; Jong-Rung Tsai; Shyng-Shiou F. Yuan; Hui-Min Wang; Wei-Lun Liu; Po-Len Liu
Journal: Mediators of Inflammation
 (2014)

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## Abstract

Acute lung injury (ALI) is associated with an inflammation-mediated process, and the transcription factor, Krüppel-like factor 5 (KLF5), might play a crucial role in inflammatory lung disease. In this study, we evaluated KLF5, reactive oxygen species (ROS), and inflammatory responses in a lipopolysaccharide- (LPS-) induced ALI model to elucidate the role of KLF5 in ALI. Our data indicated that LPS upregulates proinflammatory cytokine expression in human bronchial epithelial cells in a dose-dependent manner. We observed upregulated KLF5 protein expression in human bronchial epithelial cells exposed to LPS, with peak expression 1 h after LPS treatment, and subsequent upregulation of p65 protein expression and p65 phosphorylation at Ser276. These results indicate that KLF5 mediates proinflammatory cytokine expression by upregulating nuclear factor-kappaB (NF-κB) phosphorylation at p65 in response to LPS. LPS treatment also increased ROS production and simultaneously upregulated KLF5 expression and NF-κB translocation. N-acetylcysteine significantly reduced ROS levels and KLF5 and NF-κB translocation in nuclear extracts. Therefore, N-acetylcysteine pretreatment before LPS exposure reduces ROS, downregulates KLF5 expression, and subsequently reduces inflammatory responses by scavenging ROS. Overall, our study results indicate that KLF5 mediates proinflammatory cytokine expression through upregulation of NF-κB phosphorylation at p65 in LPS-induced ALI.


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## Body

## 1. Introduction

Acute lung injury (ALI) is a pulmonary emergency that presents with hypoxemia resistant to oxygen therapy. ALI and its more severe form, acute respiratory distress syndrome (ARDS), are associated with sudden changes in the integrity of the alveolar wall and reduced gas exchange that lead to widespread alveolar filling or collapse, which increases breathing effort and causes acute hypoxemic respiratory failure [1, 2]. ALI and ARDS are associated with increased pulmonary morbidity and mortality and increased burdens of medical care. Treatments for ALI/ARDS patients are primarily supportive and include lung protection strategies, antibiotics targeting the cause of infection, and restrictive fluid management [3–5]. No effective treatment exists for ARDS. Therefore, preventing and treating ALI/ARDS at an early stage is critical.ALI and ARDS typically develop after direct (e.g., pneumonia and pulmonary aspiration) or indirect (e.g., acute pancreatitis, massive blood transfusion, or septicemia) lung injury. The acute (or exudative) phase of ALI/ARDS is characterized by pulmonary capillary leakage with interstitial and alveolar edema and hemorrhage, surfactant dysfunction, and subsequent hyaline membrane formation [1, 3]. Studies have suggested that dysregulated recruitment of leukocytes, inappropriate expression of inflammatory cytokines, expression of eicosanoids, generation of reactive oxygen species (ROS), and uncontrolled platelet or coagulation activity play roles in the acute stages of ALI/ARDS [6, 7]. Disease progression is associated with the activation of the mitogen-activated protein kinase signaling pathway and inflammation-related transcription factors, including nuclear factor-kappaB (NF-κB), activator protein 1, and nuclear factor (erythroid-derived 2)-like 2, which regulate cell proinflammatory cytokine-associated genes to attenuate the production of interleukin- (IL-) 1β, IL-6, IL-8, tumor necrosis factor-α (TNF-α), and inducible nitric oxide synthase in lipopolysaccharide- (LPS-) induced ALI [7, 8].The Krüppel-like transcription factor (KLF) family was first identified inDrosophila in 1986, and its members (KLF1-20) contribute to several aspects of cellular growth, development, differentiation, and apoptosis. A KLF contains 3 zinc fingers at or near the C-terminus [9, 10]. KLF5 is widely expressed in various tissues, including the intestinal epithelial cells, skin, and skeletal muscle cells, and is dysregulated in several cancer cell types. It might also play an essential role in inflammatory diseases [11]. LPS induces and upregulates KLF5 expression in intestinal epithelium cells [12] and human umbilical vein endothelial cells [13]. However, few studies have evaluated the role of KLF5 in human lung inflammation associated with ALI/ARDS.The endotoxin LPS-induced model of ALI has been widely used to investigate the mechanisms of ALI. In this study, we evaluated KLF5, ROS, and inflammatory responses in an LPS-induced model of ALI using 2 normal human bronchial epithelial cell lines, HBEC and BEAS-2B, to clarify the role of KLF5 in ALI.


## 2. Materials and Methods

### 2.1. Reagents

Antibodies (Abs) for KLF5, IL-6, lamin A/C,α-tubulin, and p65 were purchased from GeneTex (Irvine, California, USA). Abs for TNF-α, phospho-p65 (Ser276), phospho-p65 (Ser536), and glyceraldehyde-3-phosphate dehydrogenase were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). 2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA), N-acetylcysteine (NAC), and LPS were obtained from Sigma (St. Louis, MO, USA). A KLF5 plasmid construct was purchased from GeneTex, and a KLF5 small-interfering RNA (siRNA) construct was purchased from Ambion (Austin, TX, USA). An electrophoretic mobility shift assay (EMSA) kit for NF-κB was obtained from Roche (Indianapolis, IN, USA), and nuclear protein extraction kits were obtained from Millipore (Temecula, CA, USA).


### 2.2. Cell Culture

The human bronchial epithelial cell lines HBEC (ScienCell, Carlsbad, CA, USA) and BEAS-2B (ATCC CRL-9609, Manassas, VA, USA) were cultured in F12 K medium (Invitrogen, NY, USA) or LHC-9 (Invitrogen) supplemented with 5% fetal bovine serum, 100 U/mL penicillin, 100μg/mL penicillin, and 100 pg/mL streptomycin (Invitrogen) in a humidified incubator with 5% CO2 at 37°C.


### 2.3. Measurement of Intracellular ROS Levels

The ROS-sensitive fluorescent dye DCFH-DA was used to determine LPS-upregulated intracellular ROS levels in cells from the HBEC and BEAS-2B lines. ROS, particularly H2O2, oxidize DCFH-DA to produce fluorescent 2,7-dichlorofluorescein [14]. For observation of intracellular ROS production through the oxidation of DCFH-DA, cells were pretreated with or without NAC (10 mM) for 1 h, washed with warm Hank’s balanced salt solution (HBSS), and incubated in HBSS or a cell medium containing 20 μM DCFH-DA at 37°C for 30 min. HBSS containing DCFH-DA was removed and replaced with fresh cell medium. The cells were then incubated with or without LPS (5 μg/mL) for 1 h, washed 3 times with phosphate-buffered saline (PBS), and detached with trypsin/ethylenediaminetetraacetic acid (EDTA). The fluorescence intensity of the cells was analyzed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA) at 485 nm excitation and 530 nm emission for 2,7-dichlorofluorescein.


### 2.4. Cytosolic and Nuclear Protein Extraction

Cells were lysed with lysis buffer (0.5 M NaCl, 50 mM Tris, 1 mM EDTA, 0.05% sodium dodecyl sulfate [SDS], 0.5% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride [PMSF]), pH 7.4, for 30 min at 4°C. Then the cell lysates were centrifuged at 4,000 ×g for 30 min at 4°C. Cytosolic protein concentrations in the supernatants were measured using a Bio-Rad protein determination kit (Bio-Rad, Hercules, CA). Nuclear protein extracts were prepared as previously described. Briefly, after washing with PBS, the cells were scraped off the plates in 0.6 mL of ice-cold buffer A consisting of 10 mM N-(2-hydroxyethyl) piperazine-N′-(2-ethenesulfonic acid), pH 7.9, 10 mM KCl, 1 mM dithiothreitol, 1 mM PMSF, 1.5 mM MgCl2, and 2 μg/mL each of aprotinin, pepstatin, and leupeptin. After centrifugation at 300 ×g for 10 min at 4°C, the cells were resuspended in buffer B (80 μL of 0.1% Triton X-100 in buffer A), left on ice for 10 min, and centrifuged at 12,000 ×g for 10 min at 4°C. The nuclear pellets were resuspended in 70 μL of ice-cold buffer C (20 mM N-[2-hydroxyethyl] piperazine-N′-[2-ethenesulfonic acid], pH 7.9, 1.5 mM MgCl2, 0.42 M NaCl, 1 mM dithiothreitol, 0.2 mM EDTA, 1 mM PMSF, 25% glycerol, and 2 μg/mL each of aprotinin, pepstatin, and leupeptin) and incubated for 30 min at 4°C, followed by centrifugation at 15,000 ×g for 30 min at 4°C. The resulting supernatant was stored at –70°C as the nuclear extract. Protein concentrations were determined using the Bio-Rad method.


### 2.5. Western Blot Analysis

Cytoplasm protein extracts or nuclear protein extracts were separated using 12% SDS polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and kept at room temperature for 1 h. The membranes were then treated with PBS containing 0.05% Tween 20 and 2% skim milk for 1 h at room temperature and incubated separately with various primary Abs followed by secondary Abs. The protein bands were detected using an enhanced chemiluminescence kit (PerkinElmer, Waltham, MA, USA) and exposure to Biomax MR film (Kodak, Rochester, NY, USA). Data were quantified using ImageQuant 5.2 (Healthcare Bio-Sciences, Philadelphia, PA, USA).


### 2.6. KLF5 Overexpression and Silencing in Human Bronchial Epithelial Cells

Cells from the HBEC line grown to 90% confluence were transfected with the pKLF5 plasmid by using Lipofectin reagent (Invitrogen). To generate cell lines expressing the various constructs, we diluted the cells, seeded then 24 h after transfection, and maintained them in F12 K medium (serum-free) for 48 h. A specific double-stranded 21-nucleotide RNA sequence homologous to the target gene, siRNA, was used to silence KLF5 expression. KLF5 siRNA primer 5′-GGU GAA CAA UAU UUU CAU CTT-3′ and 5′-GAU GAA AAU AUU GUU CAC CTC-3′.KLF5 protein expression was analyzed using western blotting after the transfection of cells with KLF5 siRNA. Briefly, cells were cultured in 100 mm dishes and transiently transfected with 20 nM siRNA using 8μL of siPORT Amine (Ambion) in a total transfection volume of 0.5 mL of medium. After incubation at 37°C in 5% CO2 for 5 h, 1.5 mL of normal growth medium was added to the cells, which were then incubated for 48 h.


### 2.7. Electrophoretic Mobility Shift Assay

The NF-phoretic mobility shift assay medium was added to the cells, which were then incubated for 48 h. 5′-ACA AGG GAC TTT CCG CTG GGG ACT TTC CAG G-3′; 3′-TGT TCC CTG AAA GGC GAC CCC TGA AAG GTC C-5′) containing a direct repeat of theκB site. The digoxigenin gel shift kit for 3′-end labeling of oligonucleotides (Roche) was used during the assay of nuclear protein-DNA binding.


### 2.8. Coimmunoprecipitation Assay

To determine the protein-protein interactions between KLF5 and NF-κB, we harvested cells from the HBEC line in immunoprecipitation lysis buffer (GeneTex). Eight hundred micrograms of nuclear protein was cleared via incubation with protein A/G agarose beads (30 μL/tube), rotated for 1 h at 4°C, and then washed. The supernatants were collected and incubated with 2 μg of anti-KLF5, anti-p-p65(Ser276), or control rabbit immunoglobulin G (IgG) Ab for 4 h. Protein A/G agarose beads (50 μL/tube) were then added to each tube and incubated overnight at 4°C. The supernatants were removed via centrifugation at 12,000 ×g for 10 min and disrupted by boiling in 1% SDS. Immune complexes were analyzed by immunoblotting with anti-KLF5, anti-p65, anti-p-p65 (Ser276), and anti-p-p65 (Ser536) Abs.


### 2.9. Mouse Model

All animal experiments were approved and supported by the Animal Center of Kaohsiung Medical University (IACUC#102007). Eight-week-oldBALB/C mice were purchased from Lasco, Taiwan. The mice were intraperitoneally injected with or without NAC (150 mg/kg) for 1 h before intraperitoneal administration of LPS (20 mg/kg) for 8 h. Control animals were injected with PBS. Seven mice were used for each treatment.Six hours after treatment, the right lungs of the mice were excised, and cytoplasmic protein was extracted and stored at –80°C for further analysis. The left lungs were isolated, placed into a tube containing 4% paraformaldehyde/PBS, and stored at 4°C for 12 h. After 3 washings, the tissues were embedded in paraffin and cut into 5 μm sections. Hematoxylin and eosin staining was used to examine the inflammatory status of the lung tissues.


### 2.10. Immunohistochemical Staining

The lung paraffin sections were deparaffinized with xylene and stained with anti-human-KLF5 Ab. After washing with PBS, the sections were incubated with horseradish peroxidase-conjugated secondary Ab for 1 h at room temperature. Diaminobenzidine was used for visualization and hematoxylin was used for counterstaining. In the negative controls, the antibody was replaced with control IgG.


### 2.11. Statistical Analyses

Results are expressed as mean ± standard error of the mean. All data were analyzed using analysis of variance and a subsequent Dunnett’s test. All statistical analyses were performed using SigmaStat version 3.5 (Systat Software Inc., Chicago, IL, USA), and aP value of <0.05 was considered statistically significant.


## 2.1. Reagents

Antibodies (Abs) for KLF5, IL-6, lamin A/C,α-tubulin, and p65 were purchased from GeneTex (Irvine, California, USA). Abs for TNF-α, phospho-p65 (Ser276), phospho-p65 (Ser536), and glyceraldehyde-3-phosphate dehydrogenase were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). 2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA), N-acetylcysteine (NAC), and LPS were obtained from Sigma (St. Louis, MO, USA). A KLF5 plasmid construct was purchased from GeneTex, and a KLF5 small-interfering RNA (siRNA) construct was purchased from Ambion (Austin, TX, USA). An electrophoretic mobility shift assay (EMSA) kit for NF-κB was obtained from Roche (Indianapolis, IN, USA), and nuclear protein extraction kits were obtained from Millipore (Temecula, CA, USA).


## 2.2. Cell Culture

The human bronchial epithelial cell lines HBEC (ScienCell, Carlsbad, CA, USA) and BEAS-2B (ATCC CRL-9609, Manassas, VA, USA) were cultured in F12 K medium (Invitrogen, NY, USA) or LHC-9 (Invitrogen) supplemented with 5% fetal bovine serum, 100 U/mL penicillin, 100μg/mL penicillin, and 100 pg/mL streptomycin (Invitrogen) in a humidified incubator with 5% CO2 at 37°C.


## 2.3. Measurement of Intracellular ROS Levels

The ROS-sensitive fluorescent dye DCFH-DA was used to determine LPS-upregulated intracellular ROS levels in cells from the HBEC and BEAS-2B lines. ROS, particularly H2O2, oxidize DCFH-DA to produce fluorescent 2,7-dichlorofluorescein [14]. For observation of intracellular ROS production through the oxidation of DCFH-DA, cells were pretreated with or without NAC (10 mM) for 1 h, washed with warm Hank’s balanced salt solution (HBSS), and incubated in HBSS or a cell medium containing 20 μM DCFH-DA at 37°C for 30 min. HBSS containing DCFH-DA was removed and replaced with fresh cell medium. The cells were then incubated with or without LPS (5 μg/mL) for 1 h, washed 3 times with phosphate-buffered saline (PBS), and detached with trypsin/ethylenediaminetetraacetic acid (EDTA). The fluorescence intensity of the cells was analyzed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA) at 485 nm excitation and 530 nm emission for 2,7-dichlorofluorescein.


## 2.4. Cytosolic and Nuclear Protein Extraction

Cells were lysed with lysis buffer (0.5 M NaCl, 50 mM Tris, 1 mM EDTA, 0.05% sodium dodecyl sulfate [SDS], 0.5% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride [PMSF]), pH 7.4, for 30 min at 4°C. Then the cell lysates were centrifuged at 4,000 ×g for 30 min at 4°C. Cytosolic protein concentrations in the supernatants were measured using a Bio-Rad protein determination kit (Bio-Rad, Hercules, CA). Nuclear protein extracts were prepared as previously described. Briefly, after washing with PBS, the cells were scraped off the plates in 0.6 mL of ice-cold buffer A consisting of 10 mM N-(2-hydroxyethyl) piperazine-N′-(2-ethenesulfonic acid), pH 7.9, 10 mM KCl, 1 mM dithiothreitol, 1 mM PMSF, 1.5 mM MgCl2, and 2 μg/mL each of aprotinin, pepstatin, and leupeptin. After centrifugation at 300 ×g for 10 min at 4°C, the cells were resuspended in buffer B (80 μL of 0.1% Triton X-100 in buffer A), left on ice for 10 min, and centrifuged at 12,000 ×g for 10 min at 4°C. The nuclear pellets were resuspended in 70 μL of ice-cold buffer C (20 mM N-[2-hydroxyethyl] piperazine-N′-[2-ethenesulfonic acid], pH 7.9, 1.5 mM MgCl2, 0.42 M NaCl, 1 mM dithiothreitol, 0.2 mM EDTA, 1 mM PMSF, 25% glycerol, and 2 μg/mL each of aprotinin, pepstatin, and leupeptin) and incubated for 30 min at 4°C, followed by centrifugation at 15,000 ×g for 30 min at 4°C. The resulting supernatant was stored at –70°C as the nuclear extract. Protein concentrations were determined using the Bio-Rad method.


## 2.5. Western Blot Analysis

Cytoplasm protein extracts or nuclear protein extracts were separated using 12% SDS polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and kept at room temperature for 1 h. The membranes were then treated with PBS containing 0.05% Tween 20 and 2% skim milk for 1 h at room temperature and incubated separately with various primary Abs followed by secondary Abs. The protein bands were detected using an enhanced chemiluminescence kit (PerkinElmer, Waltham, MA, USA) and exposure to Biomax MR film (Kodak, Rochester, NY, USA). Data were quantified using ImageQuant 5.2 (Healthcare Bio-Sciences, Philadelphia, PA, USA).


## 2.6. KLF5 Overexpression and Silencing in Human Bronchial Epithelial Cells

Cells from the HBEC line grown to 90% confluence were transfected with the pKLF5 plasmid by using Lipofectin reagent (Invitrogen). To generate cell lines expressing the various constructs, we diluted the cells, seeded then 24 h after transfection, and maintained them in F12 K medium (serum-free) for 48 h. A specific double-stranded 21-nucleotide RNA sequence homologous to the target gene, siRNA, was used to silence KLF5 expression. KLF5 siRNA primer 5′-GGU GAA CAA UAU UUU CAU CTT-3′ and 5′-GAU GAA AAU AUU GUU CAC CTC-3′.KLF5 protein expression was analyzed using western blotting after the transfection of cells with KLF5 siRNA. Briefly, cells were cultured in 100 mm dishes and transiently transfected with 20 nM siRNA using 8μL of siPORT Amine (Ambion) in a total transfection volume of 0.5 mL of medium. After incubation at 37°C in 5% CO2 for 5 h, 1.5 mL of normal growth medium was added to the cells, which were then incubated for 48 h.


## 2.7. Electrophoretic Mobility Shift Assay

The NF-phoretic mobility shift assay medium was added to the cells, which were then incubated for 48 h. 5′-ACA AGG GAC TTT CCG CTG GGG ACT TTC CAG G-3′; 3′-TGT TCC CTG AAA GGC GAC CCC TGA AAG GTC C-5′) containing a direct repeat of theκB site. The digoxigenin gel shift kit for 3′-end labeling of oligonucleotides (Roche) was used during the assay of nuclear protein-DNA binding.


## 2.8. Coimmunoprecipitation Assay

To determine the protein-protein interactions between KLF5 and NF-κB, we harvested cells from the HBEC line in immunoprecipitation lysis buffer (GeneTex). Eight hundred micrograms of nuclear protein was cleared via incubation with protein A/G agarose beads (30 μL/tube), rotated for 1 h at 4°C, and then washed. The supernatants were collected and incubated with 2 μg of anti-KLF5, anti-p-p65(Ser276), or control rabbit immunoglobulin G (IgG) Ab for 4 h. Protein A/G agarose beads (50 μL/tube) were then added to each tube and incubated overnight at 4°C. The supernatants were removed via centrifugation at 12,000 ×g for 10 min and disrupted by boiling in 1% SDS. Immune complexes were analyzed by immunoblotting with anti-KLF5, anti-p65, anti-p-p65 (Ser276), and anti-p-p65 (Ser536) Abs.


## 2.9. Mouse Model

All animal experiments were approved and supported by the Animal Center of Kaohsiung Medical University (IACUC#102007). Eight-week-oldBALB/C mice were purchased from Lasco, Taiwan. The mice were intraperitoneally injected with or without NAC (150 mg/kg) for 1 h before intraperitoneal administration of LPS (20 mg/kg) for 8 h. Control animals were injected with PBS. Seven mice were used for each treatment.Six hours after treatment, the right lungs of the mice were excised, and cytoplasmic protein was extracted and stored at –80°C for further analysis. The left lungs were isolated, placed into a tube containing 4% paraformaldehyde/PBS, and stored at 4°C for 12 h. After 3 washings, the tissues were embedded in paraffin and cut into 5 μm sections. Hematoxylin and eosin staining was used to examine the inflammatory status of the lung tissues.


## 2.10. Immunohistochemical Staining

The lung paraffin sections were deparaffinized with xylene and stained with anti-human-KLF5 Ab. After washing with PBS, the sections were incubated with horseradish peroxidase-conjugated secondary Ab for 1 h at room temperature. Diaminobenzidine was used for visualization and hematoxylin was used for counterstaining. In the negative controls, the antibody was replaced with control IgG.


## 2.11. Statistical Analyses

Results are expressed as mean ± standard error of the mean. All data were analyzed using analysis of variance and a subsequent Dunnett’s test. All statistical analyses were performed using SigmaStat version 3.5 (Systat Software Inc., Chicago, IL, USA), and aP value of <0.05 was considered statistically significant.


## 3. Results

### 3.1. LPS Upregulates Proinflammatory Cytokine Expression in Human Bronchial Epithelial Cells in a Dose-Dependent Manner

Our western blot analysis results indicated that protein expression was upregulated in a dose-dependent manner in cells from the HBEC (Figure1(a)) and BEAS-2B (Figure 1(b)) lines treated with 0, 1.25, 2.5, 5, and 10 μg/mL LPS, TNF-α, IL-1β, and IL-8.Effects of lipopolysaccharide (LPS) exposure on proinflammatory cytokine expression in human bronchial epithelial cells. Cells from the (a, b) HBEC and (c, d) BEAS-2B lines were incubated with various concentrations of LPS for various durations, and proinflammatory cytokine expression was evaluated using western blotting. Tumor necrosis factor-α (TNF-α), interleukin- (IL-) 1β, and IL-8 proteins expression were upregulated in a dose-dependent manner. Data are presented as means ± standard error of the mean (SEM) from 3 independent experiments. Each bar graph shows summarized data from 3 separate densitometry experiments after normalization to α-tubulin (an internal control for cytoplasm protein). P
*
<
0
.
05 versus LPS dose 0 or time 0.
(a)
(b)


### 3.2. LPS Upregulates KLF5 and NF-κB Subunit Expression in Human Bronchial Epithelial Cells

We observed upregulation of KLF5 and p65 expression and p65 phosphorylation at Ser276 or Ser536 to similar extents in the HBEC (Figure2(a)) and BEAS-2B (Figure 2(b)) cell lines after treatment with LPS (5 μg/mL) for various durations. However, the time to the peak intensities of expression differed slightly. We observed peak KLF5 protein expression after 1 h of LPS stimulation in both cell lines, whereas peak p65 protein expression occurred after 2 h and 1.5 h of LPS stimulation in the HBEC and BEAS-2B cell lines, respectively. We observed peak Ser276 and Ser536 phosphorylation after 1.5–2 h of LPS treatment in the HBEC and BEAS-2B cell lines.Effects of lipopolysaccharide (LPS) exposure on Krüppel-like factor 5 (KLF5) and nuclear factor-kappaB subunit protein expression in human bronchial epithelial cells. Cells from the (a) HBEC and (b) BEAS-2B lines were incubated with LPS (5μg/mL) for various durations, and KLF5, p65, phospho-p65 (Ser276), and phospho-p65 (Ser536) protein expression were evaluated using western blotting. The time to peak intensities of KLF5, p65, Ser276, and Ser536 phosphorylation differed slightly. Data were obtained from 3 independent experiments.
(a)
(b)


### 3.3. LPS-Induced Oxidative Stress Upregulates KLF5 Expression and NF-κB Subunit Translocation into the Nucleus

We pretreated the cells with or without the ROS scavenger NAC (10 mM) for 1 h and then exposed them to LPS for 1 h. Measurements of ROS with DCFH-DA assays indicated that 1 h of LPS treatment increased intracellular ROS levels in the HBEC (Figure3(a)) and BEAS-2B (Figure 3(b)) cell lines. Cells pretreated with NAC exhibited no increase in ROS levels after exposure to LPS for 1 h. Previous studies have indicated that LPS upregulates KLF5 expression and NF-κB activity by increasing the nuclear translocation of KLF5 and NF-κB. Our western blot analysis data showed that LPS increased KLF5 and NF-κB translocation in the HBEC and BEAS-2B cell lines and that pretreatment with NAC significantly reduced LPS-induced KLF5 and NF-κB translocation in nuclear extracts of cells from the HBEC (Figure 3(c)) and BEAS-2B (Figure 3(d)) lines (P
<
0
.
05).Effects of lipopolysaccharide (LPS) exposureon intracellular reactive oxygen species (ROS) levels and Krüppel-like factor 5 (KLF5) and p65 expression in human bronchial epithelial cells. Cells from the (a) HBEC and (b) BEAS-2B lines were incubated with medium alone (control), N-acetylcysteine (NAC; 10 mM), or 5μg/mL LPS for 1 h. Intracellular levels of ROS were measured using 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) assays and flow cytometric analysis. Histograms represent 3 independent experiments. Cells from the (c) HBEC and (d) BEAS-2B lines were incubated with LPS for 1.5 h, and nuclear protein expression was measured using western blotting. DCFH-DA assays indicated that 1 h of LPS treatment increased intracellular ROS levels in the HBEC (a) and BEAS-2B (b) cell lines. LPS also increased KLF5 and nuclear factor-kappaB (NF-κB) translocation in the HBEC and BEAS-2B cell lines. Pretreatment with NAC significantly reduced LPS-induced KLF5 and NF-κB translocation in nuclear extracts of cells from the HBEC (c) and BEAS-2B (d) lines. Each bar graph shows summarized data (mean ± SEM) from 3 separate densitometry experiments after normalization to lamin A/C (an internal control). P
*
<
0
.
05 versus control condition.
(a)
(b)
(c)
(d)


### 3.4. Regulatory Effects of KLF5 on p65 Phosphorylation and NF-κB Activity

According to Nihira et al. [15], phosphorylation of p65 at Ser276 prevents the degradation of p65 by ubiquitin-proteasome machinery and subsequently induces the transactivation of p65. To evaluate the effects of KLF5 on p65 phosphorylation and NF-κB activity, we transfected cells from the HBEC and BEAS-2B lines with the pKLF5 plasmid or KLF5-specific siRNA for 48 h to overexpress or silence KLF5, respectively. We then treated the cells with or without LPS for 1 h. Western blot analysis results indicated that KLF5 gene overexpression significantly increased LPS-induced p65 accumulation in the nucleus. Conversely, the silencing of KLF5 messenger RNA (mRNA) expression reduced LPS-induced p65 accumulation in the nucleus (Figure 4(a)). We observed significantly increased NF-κB binding activity in response to LPS in cells transfected with the KLF5 plasmid compared with that in the control group (medium alone), cells treated with LPS alone, or cells transfected with KLF5-specific siRNA (Figure 4(b)). We also observed significantly increased levels of the proinflammatory cytokines TNF-α, IL-1β, and IL-6 in cells transfected with the KLF5 plasmid compared with that in the control group (medium alone), cells treated with LPS alone, or cells transfected with KLF5-specific siRNA (Figure 4(c)). Cells pretreated with the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC; 100 μM) for 1 h exhibited no reduction in KLF5 protein expression after LPS exposure (Figure 4(d)).Effects ofKrüppel-like factor 5 (KLF5) overexpression or silencing on p65 phosphorylation and nuclear factor-kappaB (NF-κB) activity. (a) Cells from the HBEC line were pretreated with the pKLF5 plasmid or transfected with KLF5 small interfering RNA (siRNA) for 48 h, and KLF5, p65, phospho-p65 (Ser276), and phospho-p65 (Ser536) protein expression were measured using western blotting. (b) NF-κB activity was measured using an electrophoretic mobility shift assay, and (c) the proinflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin- (IL-) 1β, and IL-6 were evaluated using western blotting. (d) Cells from the HBEC line were pretreated with N-acetylcysteine (NAC; 10 mM) or pyrrolidine dithiocarbamate (PDTC; 100 μM) for 1 h and then treated with lipopolysaccharide (LPS; 5 μg/mL) for 1 h. KLF5 protein expression was evaluated using western blotting. Silencing of KLF5 messenger RNA (mRNA) expression reduced LPS-induced p65 accumulation in the nucleus. (a) Compared with that in the control group, cells treated with LPS alone, or cells transfected with KLF5-specific small interfering RNA (siRNA), NF-κB binding activity in cells transfected with the KLF5 plasmid increased in response to LPS. (b) Compared with those in the control group, cells treated with LPS alone, or cells transfected with KLF5-specific siRNA, levels of the proinflammatory cytokines TNF-α, IL-1β, and IL-6 in cells transfected with the KLF5 plasmid were increased. (c) Cells pretreated with the NF-κB inhibitor PDTC (100 μM) for 1 h exhibited no reduction in KLF5 protein expression after LPS exposure. (d) Data are presented as means ± SEM from 3 independent experiments. Each bar graph shows summarized data (mean ± SEM) from 3 separate densitometry experiments after normalization to glyceraldehyde-3-phosphate dehydrogenase (GAPDH; an internal control for nuclear protein). P
*
<
0
.
05 versus medium alone or control condition.
(a)
(b)
(c)
(d)


### 3.5. KLF5 Binding to NF-κB Subunits and p65 at Ser276 in LPS-Treated Human Bronchial Epithelial Cells

Subsequently, we examined whether LPS-dependent NF-κB activation is associated with upregulated KLF5 and p65 expression and phospho-p65 (Ser276) binding in cells from the HBEC line after LPS exposure. We immunoprecipitated nuclear protein lysates using anti-KLF5, anti-phospho-p65 (Ser276), or control IgG Ab and performed immunoblotting with anti-KLF5, anti-phospho-p65 (Ser276), anti-p65, and anti-phospho-p65 (Ser536) Abs. Our results demonstrated that LPS-induced KLF5 coprecipitates with KLF5 (Figure 5(a)), p65 (Figure 5(b)), and phospho-p65 (Ser276) (Figure 5(c)) but not phospho-p65 (Ser536) (Figure 5(d)). We then performed immunoprecipitation with a phospho-p65 (Ser276) antibody and immunoblotting for phospho-p65 (Ser276) (Figure 5(e)) and KLF5 (Figure 5(f)). Phospho-p65 (Ser276) interacted with KLF5 proteins in cells from the HBEC line; however, pretreatment with the ROS scavenger NAC attenuated this interaction. These results indicated that KLF5 directly and specifically interacts with p65 and phospho-p65 (Ser276) in response to LPS exposure to prevent ubiquitin-mediated degradation of the RelA subunit.Krüppel-like factor 5 (KLF5) interacts with nuclear factor-kappaB subunit in the nuclei of human bronchial epithelial cells. Cells from the HBEC line were treated with lipopolysaccharide (LPS; 5μg/mL) for 1 h, with or without N-acetylcysteine (NAC) pretreatment (10 mM). The nuclear protein extracts were immunoprecipitated with KLF5 or anti-immunoglobulin G (IgG) antibodies (Abs) and then analyzed using western blotting and Abs against (a) KLF5 (molecular weight [MW]: 50 kDa), (b) p65 (MW: 65 kDa), (c) phospho-p65 (Ser276; MW: 65 kDa), and (d) phospho-p65 (Ser536; MW: 65 kDa). Nuclear protein extracts were immunoprecipitated with Abs against phospho-p65 (Ser276) or anti-IgG and then analyzed using western blotting and Abs against (e) phospho-p65 (Ser276) and (f) KLF5 to confirm the interaction between KLF5 and phospho-p65 (Ser276). The target protein was marked along with a nonspecific binding site. LPS-induced KLF5 coprecipitates with KLF5 (a), p65 (b), and phospho-p65 (Ser276) (c) but not with phospho-p65 (Ser536) (d). The results of immunoprecipitation with a phospho-p65 (Ser276) Ab and immunoblotting for phospho-p65 (Ser276) (e) and KLF5 (f) showed that phospho-p65 (Ser276) interacted with KLF5 proteins in cells from the HBEC line.
(a)
(b)
(c)
(d)
(e)
(f)


### 3.6. KLF5, NF-κB Subunit, and Proinflammatory Cytokine Expression in LPS-Induced Acute Lung Inflammation in Mice

To evaluate KLF5 expression and histological changes in our LPS induced-ALI animal model, we compared LPS-challengedBALB/C mice (with or without NAC pretreatment) with control animals injected with saline. Hematoxylin and eosin staining of the lung sections revealed the infiltration of inflammatory cells (Figure 6(a)), and immunohistochemical analysis demonstrated upregulated KLF5 expression (Figure 6(b)) in the LPS-challenged mice.BALB/C mice challenged with LPS exhibited histological changes, including inflammatory cell infiltration, focal areas of fibrosis with collapsed air alveoli, and thickening of the alveolar wall (see Figure 6(a)) as well as upregulated KLF5, p65, phospho-p65 (Ser276), and phospho-p65 (Ser536) protein expression (Figure 6(c)) and upregulated TNF-α, IL-1β, and IL-6 expression (Figure 6(d)). NAC pretreatment significantly attenuated the LPS-induced pathological changes and inflammatory responses.Krüppel-like factor 5, nuclear factor-kappaB subunit, and proinflammatory cytokine expression in lipopolysaccharide- (LPS-) induced acute lung inflammation in mice.BALB/C mice were intraperitoneally injected with or without N-acetylcysteine (NAC; 150 mg/kg) for 1 h before intraperitoneal administration of LPS (20 mg/kg) for 8 h. Lung tissue sections were (a) stained with hematoxylin and eosin to observe the infiltration of cells and (b) analyzed immunohistochemically to evaluate KLF5 expression. A representative stained lung tissue from 3 independent experiments is shown. (c) KLF5, p65, phospho-p65 (Ser276), and phospho-p65 (Ser536) protein expression and (d) tumor necrosis factor-α (TNF-α), interleukin- (IL-) 1β, and IL-6 expression were evaluated using western blotting. We observed histological changes including inflammatory cell infiltration, focal areas of fibrosis with collapsed air alveoli, and thickening of the alveolar wall (a) as well as upregulated KLF5 (b), p65, phospho-p65 (Ser276), and phospho-p65 (Ser536) protein expression (c) and upregulated TNF-α, IL-1β, and IL-6 expression (d).
(a)
(b)
(c)
(d)


## 3.1. LPS Upregulates Proinflammatory Cytokine Expression in Human Bronchial Epithelial Cells in a Dose-Dependent Manner

Our western blot analysis results indicated that protein expression was upregulated in a dose-dependent manner in cells from the HBEC (Figure1(a)) and BEAS-2B (Figure 1(b)) lines treated with 0, 1.25, 2.5, 5, and 10 μg/mL LPS, TNF-α, IL-1β, and IL-8.Effects of lipopolysaccharide (LPS) exposure on proinflammatory cytokine expression in human bronchial epithelial cells. Cells from the (a, b) HBEC and (c, d) BEAS-2B lines were incubated with various concentrations of LPS for various durations, and proinflammatory cytokine expression was evaluated using western blotting. Tumor necrosis factor-α (TNF-α), interleukin- (IL-) 1β, and IL-8 proteins expression were upregulated in a dose-dependent manner. Data are presented as means ± standard error of the mean (SEM) from 3 independent experiments. Each bar graph shows summarized data from 3 separate densitometry experiments after normalization to α-tubulin (an internal control for cytoplasm protein). P
*
<
0
.
05 versus LPS dose 0 or time 0.
(a)
(b)


## 3.2. LPS Upregulates KLF5 and NF-κB Subunit Expression in Human Bronchial Epithelial Cells

We observed upregulation of KLF5 and p65 expression and p65 phosphorylation at Ser276 or Ser536 to similar extents in the HBEC (Figure2(a)) and BEAS-2B (Figure 2(b)) cell lines after treatment with LPS (5 μg/mL) for various durations. However, the time to the peak intensities of expression differed slightly. We observed peak KLF5 protein expression after 1 h of LPS stimulation in both cell lines, whereas peak p65 protein expression occurred after 2 h and 1.5 h of LPS stimulation in the HBEC and BEAS-2B cell lines, respectively. We observed peak Ser276 and Ser536 phosphorylation after 1.5–2 h of LPS treatment in the HBEC and BEAS-2B cell lines.Effects of lipopolysaccharide (LPS) exposure on Krüppel-like factor 5 (KLF5) and nuclear factor-kappaB subunit protein expression in human bronchial epithelial cells. Cells from the (a) HBEC and (b) BEAS-2B lines were incubated with LPS (5μg/mL) for various durations, and KLF5, p65, phospho-p65 (Ser276), and phospho-p65 (Ser536) protein expression were evaluated using western blotting. The time to peak intensities of KLF5, p65, Ser276, and Ser536 phosphorylation differed slightly. Data were obtained from 3 independent experiments.
(a)
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## 3.3. LPS-Induced Oxidative Stress Upregulates KLF5 Expression and NF-κB Subunit Translocation into the Nucleus

We pretreated the cells with or without the ROS scavenger NAC (10 mM) for 1 h and then exposed them to LPS for 1 h. Measurements of ROS with DCFH-DA assays indicated that 1 h of LPS treatment increased intracellular ROS levels in the HBEC (Figure3(a)) and BEAS-2B (Figure 3(b)) cell lines. Cells pretreated with NAC exhibited no increase in ROS levels after exposure to LPS for 1 h. Previous studies have indicated that LPS upregulates KLF5 expression and NF-κB activity by increasing the nuclear translocation of KLF5 and NF-κB. Our western blot analysis data showed that LPS increased KLF5 and NF-κB translocation in the HBEC and BEAS-2B cell lines and that pretreatment with NAC significantly reduced LPS-induced KLF5 and NF-κB translocation in nuclear extracts of cells from the HBEC (Figure 3(c)) and BEAS-2B (Figure 3(d)) lines (P
<
0
.
05).Effects of lipopolysaccharide (LPS) exposureon intracellular reactive oxygen species (ROS) levels and Krüppel-like factor 5 (KLF5) and p65 expression in human bronchial epithelial cells. Cells from the (a) HBEC and (b) BEAS-2B lines were incubated with medium alone (control), N-acetylcysteine (NAC; 10 mM), or 5μg/mL LPS for 1 h. Intracellular levels of ROS were measured using 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) assays and flow cytometric analysis. Histograms represent 3 independent experiments. Cells from the (c) HBEC and (d) BEAS-2B lines were incubated with LPS for 1.5 h, and nuclear protein expression was measured using western blotting. DCFH-DA assays indicated that 1 h of LPS treatment increased intracellular ROS levels in the HBEC (a) and BEAS-2B (b) cell lines. LPS also increased KLF5 and nuclear factor-kappaB (NF-κB) translocation in the HBEC and BEAS-2B cell lines. Pretreatment with NAC significantly reduced LPS-induced KLF5 and NF-κB translocation in nuclear extracts of cells from the HBEC (c) and BEAS-2B (d) lines. Each bar graph shows summarized data (mean ± SEM) from 3 separate densitometry experiments after normalization to lamin A/C (an internal control). P
*
<
0
.
05 versus control condition.
(a)
(b)
(c)
(d)


## 3.4. Regulatory Effects of KLF5 on p65 Phosphorylation and NF-κB Activity

According to Nihira et al. [15], phosphorylation of p65 at Ser276 prevents the degradation of p65 by ubiquitin-proteasome machinery and subsequently induces the transactivation of p65. To evaluate the effects of KLF5 on p65 phosphorylation and NF-κB activity, we transfected cells from the HBEC and BEAS-2B lines with the pKLF5 plasmid or KLF5-specific siRNA for 48 h to overexpress or silence KLF5, respectively. We then treated the cells with or without LPS for 1 h. Western blot analysis results indicated that KLF5 gene overexpression significantly increased LPS-induced p65 accumulation in the nucleus. Conversely, the silencing of KLF5 messenger RNA (mRNA) expression reduced LPS-induced p65 accumulation in the nucleus (Figure 4(a)). We observed significantly increased NF-κB binding activity in response to LPS in cells transfected with the KLF5 plasmid compared with that in the control group (medium alone), cells treated with LPS alone, or cells transfected with KLF5-specific siRNA (Figure 4(b)). We also observed significantly increased levels of the proinflammatory cytokines TNF-α, IL-1β, and IL-6 in cells transfected with the KLF5 plasmid compared with that in the control group (medium alone), cells treated with LPS alone, or cells transfected with KLF5-specific siRNA (Figure 4(c)). Cells pretreated with the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC; 100 μM) for 1 h exhibited no reduction in KLF5 protein expression after LPS exposure (Figure 4(d)).Effects ofKrüppel-like factor 5 (KLF5) overexpression or silencing on p65 phosphorylation and nuclear factor-kappaB (NF-κB) activity. (a) Cells from the HBEC line were pretreated with the pKLF5 plasmid or transfected with KLF5 small interfering RNA (siRNA) for 48 h, and KLF5, p65, phospho-p65 (Ser276), and phospho-p65 (Ser536) protein expression were measured using western blotting. (b) NF-κB activity was measured using an electrophoretic mobility shift assay, and (c) the proinflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin- (IL-) 1β, and IL-6 were evaluated using western blotting. (d) Cells from the HBEC line were pretreated with N-acetylcysteine (NAC; 10 mM) or pyrrolidine dithiocarbamate (PDTC; 100 μM) for 1 h and then treated with lipopolysaccharide (LPS; 5 μg/mL) for 1 h. KLF5 protein expression was evaluated using western blotting. Silencing of KLF5 messenger RNA (mRNA) expression reduced LPS-induced p65 accumulation in the nucleus. (a) Compared with that in the control group, cells treated with LPS alone, or cells transfected with KLF5-specific small interfering RNA (siRNA), NF-κB binding activity in cells transfected with the KLF5 plasmid increased in response to LPS. (b) Compared with those in the control group, cells treated with LPS alone, or cells transfected with KLF5-specific siRNA, levels of the proinflammatory cytokines TNF-α, IL-1β, and IL-6 in cells transfected with the KLF5 plasmid were increased. (c) Cells pretreated with the NF-κB inhibitor PDTC (100 μM) for 1 h exhibited no reduction in KLF5 protein expression after LPS exposure. (d) Data are presented as means ± SEM from 3 independent experiments. Each bar graph shows summarized data (mean ± SEM) from 3 separate densitometry experiments after normalization to glyceraldehyde-3-phosphate dehydrogenase (GAPDH; an internal control for nuclear protein). P
*
<
0
.
05 versus medium alone or control condition.
(a)
(b)
(c)
(d)


## 3.5. KLF5 Binding to NF-κB Subunits and p65 at Ser276 in LPS-Treated Human Bronchial Epithelial Cells

Subsequently, we examined whether LPS-dependent NF-κB activation is associated with upregulated KLF5 and p65 expression and phospho-p65 (Ser276) binding in cells from the HBEC line after LPS exposure. We immunoprecipitated nuclear protein lysates using anti-KLF5, anti-phospho-p65 (Ser276), or control IgG Ab and performed immunoblotting with anti-KLF5, anti-phospho-p65 (Ser276), anti-p65, and anti-phospho-p65 (Ser536) Abs. Our results demonstrated that LPS-induced KLF5 coprecipitates with KLF5 (Figure 5(a)), p65 (Figure 5(b)), and phospho-p65 (Ser276) (Figure 5(c)) but not phospho-p65 (Ser536) (Figure 5(d)). We then performed immunoprecipitation with a phospho-p65 (Ser276) antibody and immunoblotting for phospho-p65 (Ser276) (Figure 5(e)) and KLF5 (Figure 5(f)). Phospho-p65 (Ser276) interacted with KLF5 proteins in cells from the HBEC line; however, pretreatment with the ROS scavenger NAC attenuated this interaction. These results indicated that KLF5 directly and specifically interacts with p65 and phospho-p65 (Ser276) in response to LPS exposure to prevent ubiquitin-mediated degradation of the RelA subunit.Krüppel-like factor 5 (KLF5) interacts with nuclear factor-kappaB subunit in the nuclei of human bronchial epithelial cells. Cells from the HBEC line were treated with lipopolysaccharide (LPS; 5μg/mL) for 1 h, with or without N-acetylcysteine (NAC) pretreatment (10 mM). The nuclear protein extracts were immunoprecipitated with KLF5 or anti-immunoglobulin G (IgG) antibodies (Abs) and then analyzed using western blotting and Abs against (a) KLF5 (molecular weight [MW]: 50 kDa), (b) p65 (MW: 65 kDa), (c) phospho-p65 (Ser276; MW: 65 kDa), and (d) phospho-p65 (Ser536; MW: 65 kDa). Nuclear protein extracts were immunoprecipitated with Abs against phospho-p65 (Ser276) or anti-IgG and then analyzed using western blotting and Abs against (e) phospho-p65 (Ser276) and (f) KLF5 to confirm the interaction between KLF5 and phospho-p65 (Ser276). The target protein was marked along with a nonspecific binding site. LPS-induced KLF5 coprecipitates with KLF5 (a), p65 (b), and phospho-p65 (Ser276) (c) but not with phospho-p65 (Ser536) (d). The results of immunoprecipitation with a phospho-p65 (Ser276) Ab and immunoblotting for phospho-p65 (Ser276) (e) and KLF5 (f) showed that phospho-p65 (Ser276) interacted with KLF5 proteins in cells from the HBEC line.
(a)
(b)
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(d)
(e)
(f)


## 3.6. KLF5, NF-κB Subunit, and Proinflammatory Cytokine Expression in LPS-Induced Acute Lung Inflammation in Mice

To evaluate KLF5 expression and histological changes in our LPS induced-ALI animal model, we compared LPS-challengedBALB/C mice (with or without NAC pretreatment) with control animals injected with saline. Hematoxylin and eosin staining of the lung sections revealed the infiltration of inflammatory cells (Figure 6(a)), and immunohistochemical analysis demonstrated upregulated KLF5 expression (Figure 6(b)) in the LPS-challenged mice.BALB/C mice challenged with LPS exhibited histological changes, including inflammatory cell infiltration, focal areas of fibrosis with collapsed air alveoli, and thickening of the alveolar wall (see Figure 6(a)) as well as upregulated KLF5, p65, phospho-p65 (Ser276), and phospho-p65 (Ser536) protein expression (Figure 6(c)) and upregulated TNF-α, IL-1β, and IL-6 expression (Figure 6(d)). NAC pretreatment significantly attenuated the LPS-induced pathological changes and inflammatory responses.Krüppel-like factor 5, nuclear factor-kappaB subunit, and proinflammatory cytokine expression in lipopolysaccharide- (LPS-) induced acute lung inflammation in mice.BALB/C mice were intraperitoneally injected with or without N-acetylcysteine (NAC; 150 mg/kg) for 1 h before intraperitoneal administration of LPS (20 mg/kg) for 8 h. Lung tissue sections were (a) stained with hematoxylin and eosin to observe the infiltration of cells and (b) analyzed immunohistochemically to evaluate KLF5 expression. A representative stained lung tissue from 3 independent experiments is shown. (c) KLF5, p65, phospho-p65 (Ser276), and phospho-p65 (Ser536) protein expression and (d) tumor necrosis factor-α (TNF-α), interleukin- (IL-) 1β, and IL-6 expression were evaluated using western blotting. We observed histological changes including inflammatory cell infiltration, focal areas of fibrosis with collapsed air alveoli, and thickening of the alveolar wall (a) as well as upregulated KLF5 (b), p65, phospho-p65 (Ser276), and phospho-p65 (Ser536) protein expression (c) and upregulated TNF-α, IL-1β, and IL-6 expression (d).
(a)
(b)
(c)
(d)


## 4. Discussion

In this study, we observed upregulated KLF5 expression, ROS levels, and inflammatory responses in human bronchial epithelial cell lines and in an LPS-induced model of ALI. We also observed increased KLF5-mediated proinflammatory cytokine expression through upregulation of NF-κB phosphorylation. LPS, a component of the outer membrane of gram-negative bacteria, provokes strong immune reactions in animals and it has been widely used to study the mechanisms of ALI. LPS can induce the infiltration of inflammatory cells and the overproduction of inflammatory mediators. Our study results indicate that LPS upregulates the proinflammatory cytokines TNF-α, IL-1β, and IL-6 in the HBEC and BEAS-2B cell lines in a dose-dependent manner (see Figures 1(a) and 1(b)). We observed typical histological changes in our LPS-induced model of ALI, including increased inflammatory cell infiltration, focal areas of fibrosis with collapsed air alveoli, thickening of the alveolar wall in lung tissue, and increased expression of the proinflammatory cytokines TNF-α, IL-1β, and IL-6 (see Figures 6(a) and 6(d)).The mechanisms underlying LPS induction of inflammatory mediator overproduction have been well described. Previous studies have shown that LPS-induced expression of proinflammatory genes is associated with the nuclear transcription factor NF-κB, which plays a crucial role in mediating intracellular signaling [16–18]. The prototypical complex of NF-κB is a heterodimer of RelA/p65 and p50 subunits. Phosphorylation of Ser276 and Ser536 is essential for p65 NF-κB subunit-dependent cellular responses [19, 20]. Chanchevalap et al. [12] discovered that KLF5 plays an essential role in mediating LPS-induced proinflammatory responses in intestinal epithelial cells. Our study results indicate that LPS treatment upregulates KLF5 protein expression in human bronchial epithelial cells, with peak expression 1 h after LPS treatment. Subsequently, we observed peak p65 protein expression (after 1.5 h and 2 h in the BEAS-2B and HBEC cell lines, resp.) and peak phosphorylation of p65 at Ser276 and Ser536 (after 1.5–2 h in the HBEC and BEAS-2B cell lines; see Figures 2(a) and 2(b)). These timing sequences indicate that LPS stimulation upregulates KLF5 expression, followed by p65 expression, and then phosphorylation of p65 at Ser276 and Ser536, suggesting that KLF5 is an upstream mediator of p65 expression and phosphorylation.ROS are partially reduced metabolites of oxygen produced during normal cellular metabolism, and they have strong oxidizing capabilities. ROS are key signaling molecules in inflammatory disorders [21]. Oxidative stress is a harmful process that leads to airway and lung damage and, consequently, to severe respiratory diseases [22]. ROS induce the expression of the genes of inflammatory mediators, such as TNF-α and IL-1β [23, 24], in bronchial epithelial cells and can eventually cause pulmonary epithelial dysfunction [8, 25].ROS are considered mediators of ALI/ARDS pathogenesis. Previous studies have investigated the use of antioxidants to reduce ROS and limit the progression of ALI/ARDS. Several studies have evaluated the efficacy of the free radical scavenger NAC for reducing oxidative stress [26–29]. NAC can protect alveolar epithelial cells from H2O2-induced apoptosis by scavenging ROS [30]. Our DCFH-DA assay and western blot analysis data indicate that LPS stimulation increases ROS levels and simultaneously upregulates KLF5 expression and NF-κB translocation in cells from the HBEC and BEAS-2B lines treated with LPS compared with control cells (see Figure 3). The ROS scavenger NAC significantly reduced LPS-induced KLF5 expression and NF-κB translocation in nuclear extracts of both cells lines. NAC pretreatment significantly attenuated LPS-induced pathological changes in lung tissues ofBALB/C mice intraperitoneally injected with LPS. These results indicated associations among KLF5 expression, ROS levels, and inflammatory responses in our LPS-induced model of ALI. NAC pretreatment before LPS exposure reduced ROS levels and then downregulated KLF5 expression and subsequent inflammatory responses by scavenging ROS.KLF5 directly regulates several target genes involved in cell proliferation, the cell cycle, inflammation, and apoptosis [11]. Previous studies have shown that TNF-α and LPS upregulate KLF5 expression in intestinal epithelial cells and human umbilical vein endothelial cells [12, 13]. KLF5 directly interacts with NF-κB in epidermal epithelial cells [31]. We transfected cells with a pKLF5 plasmid or KLF5-specific siRNA to overexpress or silence KLF5, respectively, to evaluate the interaction between KLF5 and NF-κB. We observed markedly increased NF-κB binding activity in response to LPS in cells transfected with the KLF5 plasmid and significantly reduced NF-κB binding activity in cells transfected with KLF5-specific siRNA (see Figure 4(b)). According to Chanchevalap et al. [12], siRNA knockdown of KLF5 mRNA reduces the expression of the p50 and p65 subunits of NF-κB and its downstream inflammatory genes in response to LPS.Furthermore, we examined whether KLF5 plays a role in LPS-induced upregulation of TNF-α, IL-1β, and IL-6 through the NF-κB signaling pathway. Our results revealed that the silencing of KLF5 mRNA expression reduced LPS-induced p65 nuclear accumulation, indicating that KLF5 expression increases p65 nuclear localization. When we pretreated cells with the NF-κB inhibitor PDTC, nuclear KLF5 protein expression was not reduced in LPS-treated cells, suggesting that the transcription factor KLF5 translocates into the nucleus in response to LPS. Our coimmunoprecipitation assay data indicated increased nuclear KLF accumulation after LPS treatment. We observed increased binding of KLF5 with p65 and phospho-p65 (Ser276), but not with phospho-p65 (Ser536), in cells from the HBEC line after exposure to LPS (see Figures 5(a)–5(d)). Therefore, LPS-dependent NF-κB activation is associated with increased p65 and KLF5 binding. NAC attenuates nuclear KLF5 accumulation by reducing ROS in response to LPS. KLF5, p65, phospho-p65 (Ser276), and phospho-p65 (Ser536) expression were upregulated inBALB/C mice intraperitoneally injected with LPS (see Figure 6(c)).Overall, these results indicate that KLF5 is essential for the induction of NF-κB subunit expression through phosphorylation of p65 at Ser276 in response to LPS and that an ROS scavenger can reduce this response. The NF-κB inhibitor PDTC failed to inhibit KLF5 protein upregulation (see Figure 4(d)) after LPS exposure. Therefore, KLF5 is located upstream of NF-κB in the LPS-induced signaling pathway.Our results indicate that the pathway of ALI/ARDS induction is influenced by ROS production in our models. In addition, NAC pretreatment before LPS exposure reduces ROS. These findings imply that ROS scavengers might offer promising treatments for patients with ALI/ARDS in clinical settings. However, several clinical trials of NAC therapy in patients with ALI/ARDS have not shown promising outcomes [27, 28, 32]. Another approach will be novel agents developed from a better understanding of ALI/ARDS.Although we have demonstrated the pathway of KLF5-mediated proinflammatory cytokine expression through ROS and upregulation of NF-κB phosphorylation on p65 in response to LPS in human bronchial epithelial cell lines, there might be a pathway other than ROS production that precedes the inflammatory reaction in response to LPS. KLF5 itself might be moved directly to nucleus without NF-κB phosphorylation on p65. Further study is necessary to clarify the roles of KLF5 in the mediation of inflammatory reactions other than ROS production in response to LPS.


## 5. Conclusion

In summary, our study results indicate that the transcription factor KLF5 mediates proinflammatory cytokine expression through upregulation of NF-κB phosphorylation at p65in vitro andin vivo in an LPS-induced model of ALI (Figure 7). Although the NF-κB pathway is the major signaling pathway for the activation of proinflammatory gene expression in response to LPS [19, 20, 22], our results demonstrate that KLF5 also exerts a direct effect on the transcriptional activation of proinflammatory genes.Figure 7
Krüppel-like factor 5 mediation of proinflammatory cytokine expression through upregulation of nuclear factor-kappaB subunit phosphorylation at p65 in response to lipopolysaccharide in human bronchial epithelial cells.



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*Source: 281984-2014-08-12.xml*