Dexamethasone inhibits inflammatory response via down regulation of AP-1 transcription factor in human lung epithelial cells

Accepted Manuscript Dexamethasone inhibits inflammatory response via down regulation of AP-1 transcription factor in human lung epithelial cells

Rajeshwari H. Patil, M. Naveen Kumar, K.M. Kiran Kumar, Rashmi Nagesh, K. Kavya, R.L. Babu, Govindarajan T. Ramesh, S. Chidananda Sharma PII: DOI: Reference:

S0378-1119(17)31079-X https://doi.org/10.1016/j.gene.2017.12.024 GENE 42410

To appear in:

Gene

Received date: Revised date: Accepted date:

20 January 2017 12 December 2017 13 December 2017

Please cite this article as: Rajeshwari H. Patil, M. Naveen Kumar, K.M. Kiran Kumar, Rashmi Nagesh, K. Kavya, R.L. Babu, Govindarajan T. Ramesh, S. Chidananda Sharma , Dexamethasone inhibits inflammatory response via down regulation of AP-1 transcription factor in human lung epithelial cells. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Gene(2017), https://doi.org/10.1016/j.gene.2017.12.024

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ACCEPTED MANUSCRIPT Dexamethasone inhibits inflammatory response via down regulation of AP-1 transcription factor in human lung epithelial cells

Rajeshwari H. Patil*,1,4, Naveen Kumar M.1, Kiran Kumar K. M.1, Rashmi Nagesh1, Kavya K.1, Babu R. L2,3, Govindarajan T. Ramesh3 and S. Chidananda Sharma1,$

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Department of Microbiology and Biotechnology, Bangalore University, Jnana Bharathi, Bengaluru-560 056, Karnataka, India

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Department of Bioinformatics and Biotechnology, Karnataka State Women’s University, Jnana Shakthi Campus, Vijayapura, 586 108, Karnataka, India

Department of Biology and Center for Biotechnology and Biomedical Sciences, Norfolk State University, Norfolk, VA, USA

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Department of Biotechnology, The Oxford College of Science, HSR Layout, Bengaluru-560102, Karnataka, India

*Corresponding Author Dr. Rajeshwari H. Patil

Department of Microbiology and Biotechnology, Bangalore University, Bengaluru-560 056, Karnataka, India, Mobile: +91-9482224923 E-mail: [email protected]

ACCEPTED MANUSCRIPT ABSTRACT: The production of inflammatory mediators by epithelial cells in inflammatory lung diseases may represent an important target for the anti-inflammatory effects of glucocorticoids. Activator protein-1 (Khanapure et al.) is a major activator of inflammatory genes and has been proposed as a target for inhibition by glucocorticoids. We have used human pulmonary type-II A549 cells to examine the effect of dexamethasone on the phorbol ester (PMA) / Lipopolysaccharide (LPS) induced pro-inflammatory cytokines and AP-1 factors. A549 cells were treated with and

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without PMA or LPS or dexamethasone and the cell viability and nitric oxide production was measured by MTT

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assay and Griess reagent respectively. Expression of pro-inflammatory cytokines and AP-1 factors mRNAs were

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measured using semi quantitative RT-PCR. The PMA/LPS treated cells show significant 2-3 fold increase in the mRNA levels of pro-inflammatory cytokines (IL-1β, IL-2, IL-6, IL-8 and TNF-α), cyclo-oxygenase-2 (COX-2) and

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specific AP-1 factors (c-Jun, c-Fos and Jun-D). Whereas, pretreatment of cells with dexamethasone significantly inhibited the LPS induced nitric oxide production and PMA/LPS induced mRNAs expression of above pro-

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inflammatory cytokines, COX-2 and AP-1 factors. Cells treated with dexamethasone alone at both the concentrations inhibit the mRNAs expression of IL-1β, IL-6 and TNF-α compared to control. Our study reveals that

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dexamethasone decreased the mRNAs expression of c-Jun and c-Fos available for AP-1 formation suggested that AP-1 is the probable key transcription factor involved in the anti-inflammatory activity of dexamethasone. This may

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be an important molecular mechanism of steroid action in asthma and other chronic inflammatory lung diseases

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which may be useful for treatment of lung inflammatory diseases.

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Highlights:-

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Key words:-AP-1 factors; A549; Inflammation; Dexamethasone; cytokines.

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Both PMA and LPS induce the expression of inflammatory mediators in A549 cells

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Dexamethasone inhibits the PMA/LPS induced pro-inflammatory cytokines mRNA expression.

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Dexamethasone inhibits the LPS induced nitric oxide production.

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Dexamethasone decreased the PMA induced mRNAs expression of c-Jun and c-Fos AP-1.

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The anti-inflammatory activity of dexamethasone is through c-Jun and c-Fos AP-1 factor.

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AP-1 is well-described transcription factor for the target of classic GR-mediated transrepression.

ACCEPTED MANUSCRIPT Abbreviations: AP-1

Activator protein

COX-2 Cyclooxygenase-2 LPS

Lipopolysaccharide

NF- B Nuclear factor-kappa-B Nitric oxide

PMA

Phorbol-12-myristate-13-acetate

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Interleukins

TNF-α

Tumor necrosis factor - α

GREs

Glucocorticoid responsive elements

GCs

Glucocorticoids

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granulocyte macrophage colony-stimulating factors

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GM-CSF

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1. Introduction

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Inflammation is a response to tissue injury or infection, and it is characterized in its acute phase by increase in vascular permeability by plasma extravasations, resulting in an accumulation of fluid, leucocytes and mediators to

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the inflammatory site (Guo et al., 2012). Inflammation attracts immune cells and induces the local production of several cytokines (e.g. TNF-α) and chemokines (e.g. IL-8) (Rickard and Young, 2009). Expression and over

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production of pro-inflammatory cytokines promote systemic inflammation, leading to the development of many

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inflammatory diseases, such as asthma, rheumatoid arthritis and bowel diseases (Choy, 2012). For the past half a century, synthetic glucocorticoids (GCs) have been extensively used to treat chronic inflammatory diseases (Barnes,

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2006; Hillier, 2007). Glucocorticoids are among the most widely prescribed and most effective anti-inflammatory medications that are currently available. The anti-inflammatory effects of glucocorticoids are due to their ability to repress the inflammatory gene expression that occurs in all kinds of inflammatory contexts. By reducing the expression of cytokines, chemokines, adhesion molecules and other inflammatory proteins, glucocorticoids prevent the recruitment of inflammatory cells to sites of inflammation (Newton et al., 2010). Furthermore, glucocorticoids promote the cell death of many inflammatory cells and this may further contribute to reducing the inflammatory cell burden. Glucocorticoids inhibit the expression of inflammatory mediators in macrophages and other cells and are used in the treatment of many immune-mediated inflammatory diseases (Newton, 2000). Immunosuppressive

ACCEPTED MANUSCRIPT properties and their potent ability to reduce inflammation make synthetic glucocorticoids (GCs) the most prescribed drugs used in the treatment of disorders, such as asthma, arthritis or dermatitis. GCs are also used to treat patients suffering from a wide range of hematological and non-hematological cancers either because of their inhibiting effects on cell cycle progression and apoptosis promotion or for their beneficial properties, e.g. decreasing oedema, pain, nausea and reducing toxicity of the standard chemotherapy regimens in healthy tissues (Rutz, 2002; Rutz and Herr, 2004).

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The regulation of gene expression by glucocorticoids can be mediated either by the canonical mechanism that

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involves the interaction of the glucocorticoid receptor (GR), a transcription factor activated by the hormone, with

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glucocorticoid response element (Ye et al.), or by the non-canonical mechanism that involves the interaction of GR with other transcription factors, in particular nuclear factor-B (NF- B) and activator protein-1 (AP-1) complex

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(Kassel and Herrlich, 2007). Both NF-B and AP-1 are known to play an important role in gene regulation during inflammatory reaction and to regulate the transcription of inflammatory cytokines, MMPs and cyclo-oxygenase-2

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(COX-2) (Crofford et al., 1997; Bondeson et al., 2000).

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AP-1 is a dimeric transcription factor comprising of proteins derived from two super families the Jun (c-Jun, Jun-B and Jun-D) and the Fos (c-Fos, Fos-B, Fra-1 and Fra-2) proteins and is an important regulator of gene expression.

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The main AP-1 proteins in mammalian cells are Fos and Jun, which form hetero- (Jun–Fos or Jun-Fra) or homo

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dimers (Jun– Jun) complex (Angel and Karin, 1991; Shaulian and Karin, 2001). The number of combination of Jun and Fos creates an enormous functional diversity with individual AP-1 proteins probably engaged in different cell

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functions (Hess et al., 2004). Moreover, protein components of the AP-1 complex are directly involved in protein– protein interactions between various transcriptions factors (Turpaev, 2006). The AP-1 complex is a ‘signal

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converter’(Wisdom, 1999), which mediates responses to cellular signals by binding DNA and producing changes in gene transcription that ultimately lead to physiologic changes in the cell. Various Fos and Jun proteins interact with the promoters of cytokine genes either individually as AP-1 dimers, or in cooperation with other transcription factors such as NF-B, NFAT, CREB/activating transcription factor, etc.(Mechta-Grigoriou et al., 2001; Chang et al., 2002). Studies show that, the regulation of IL-4, IL-5, and GM-CSF requires the formation of NFAT/AP-1 complexes in T cells (Lee et al., 1995; Rooney et al., 1995). Similarly, in LPS-stimulated THP-1 cells, c-Juncontaining complexes have been shown to interact with NF-B proteins p50/p65, and synergistically enhance the TNF- promoter activity (Yao et al., 1997). More recently an increase in c-Fos expression and possibly AP-1

ACCEPTED MANUSCRIPT activity was described in human nasal polyps in vivo (Baraniuk et al., 1998). Hence the inhibition of AP-1 is a promising approach for the treatment of inflammation. In most of the cells, dexamethasone has been shown to inhibit NF-B and AP-1 DNA binding activity (Mukaida et al., 1994; Matsumura et al., 2001). Studies reported that dexamethasone inhibits IL-12p40 production in LPS-stimulated human monocytic cells by down-regulating the activation of JNK MAPK, the AP-1, and NF-B transcription factors (Ma et al., 2004). Although literatures are available that dexamethasone reduces inflammatory cytokines via AP-1 mechanism but, which families of AP-1

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factors are involved in anti-inflammatory activity of dexamethasone has not been done in lung epithelial cells. Type

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II alveolar epithelial cells (AECII) are since long recognized as important players of the innate immune system,

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producing the cytokines and chemokines (Corbière et al., 2011) which represents an important site of glucocorticoid action. Hence in the present work dexamethasone an agonist of glucocorticoid was used to study their anti-

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inflammatory activity towards PMA/LPS induced expression of inflammatory mediators and AP-1 factors using A549 cells. The study provides insights into the role of molecular mechanism involved in the anti-inflammatory

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activity of dexamethasone, and may help in identification of a target for therapeutic intervention.

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2. Materials and methods

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Human lung adenocarcinoma A549 cells were purchased from NCCS (Pune, India), Phorbol 12-myristate 13-acetate (PMA), LPS (Escherichia coli serotype O55:B5), Dexamethasone, Griess reagent, TRIzol, Oligos forward and

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reverse primers for different cytokines, AP-1 factors, Cyclooxygenase-2 and β-actin were designed (Patil et al., 2015), and purchased from Sigma-Aldrich (St Louis, USA). Roswell Park Memorial Institute 1640 (RPMI 1640)

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medium, Fetal bovine serum (FBS), penicillin, streptomycin, glutamine, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), trypan blue, agarose and Ethidium bromide were

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purchased from Himedia (Mumbai, India). Superscript reverse transcriptase for semi-quantitative RT-PCR was from Invitrogen (CA, USA). Taq DNA polymerase (1 U/μl) was procured from Merck-Millipore (Mumbai, India).

ACCEPTED MANUSCRIPT 2.1. Culturing of A549 cells A549 cells were grown in 25 cm2 culture flask using RPMI 1640 medium with 10 % FBS, 100 U/ml penicillin and 100µg/ml streptomycin and 2 mM L-glutamine. Cells were cultured in a humidified atmosphere at 37 °C by passing 5 % CO2 in an incubator. Flask containing 90–100 % confluent cells were sub-cultured in 96-wells plate (3x103 cells/ well) or in 6-wells plate (3x105cells/well) for the treatment and expression studies (Patil et al., 2016). (Wherever dexamethasone was used the cells were pre incubated 1 hour either with two different concentrations of

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dexamethasone (0.1 or 1 µM) and then treated with PMA (10nM) / LPS (1µg/ml) for additional 24h).

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2.2. Cell Viability assay

Analysis of cell viability was carried out using MTT assay as per the protocol described earlier (Babu et al., 2013)

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MTT is a pale yellow substrate taken up by live cells and reduced in mitochondria by Succinate dehydrogenase to yield a dark blue formazan product. A549 cells (3×103cells /well) in 200 µl of RPMI-1640 medium were seeded into

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96-wells culture plate and incubated overnight at 37 °C with the supply of 5% CO 2. Cells were treated with or without different concentrations of dexamethasone and incubated for 24 h. The cells were washed with PBS, and

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treated with 20 μl of MTT (5mg/ml) and incubated for 4 h at 37 °C in a CO2 incubator. The blue formazan products

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formed in cells were dissolved in DMSO (100μl) and spectrophotometrically measured at 540 nm. The effect of

2.3. Griess Nitrite Assay.

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dexamethasone on cell viability was calculated and represented graphically as % of viable cells compared to control.

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The NO production was measured by Griess assay as described by (Rajeshwari H. Patil. et al., 2015). Briefly, (5×105cells/well) were seeded in to 6 wells plate. After 24 h, the cells were washed with fresh medium and treated

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with and without PMA (10nM) or LPS (1μg/ml) alone or PMA with dexamethasone or LPS with dexamethasone (0.1 µM or 1 µM) for 24 h. The concentration of nitrite in the cells free culture medium was measured by adding 100μl of Griess reagent [0.1% napthylethylenediamine in dH 2O and 1% sulfanilamide in 5% (v/v) phosphoric acid, mixed 1:1 immediately before use] to 100μl of culture supernatant and incubated at room temperature for 10 min. The absorbance at 540 nm was measured using a microplate reader (PerkinElmer multimode plate reader (MA, USA). Nitrite concentration present in the spent medium was calculated using sodium nitrite as standard.

ACCEPTED MANUSCRIPT 2.4. RNA isolation and semi-quantitative RT-PCR analysis Overnight cultures of A549 cells (5×105cells/well) in a six-well plate were incubated with or without PMA (10 nM) or LPS (1μg/ml) or PMA with dexamethasone (0.1 µM or 1 µM) or LPS with dexamethasone (0.1 µM or 1 µM) or dexamethasone alone (0.1 or 1 µM) for 24 h. The concentration of PMA (10 nM) and LPS (1μg/ml) are selected based on the cell viability results of our previous study (Patil et al., 2015, Patil et al., 2016) and same concentrations were used in further experiments. Total RNA was isolated from control, and treated cells using TRIzol reagent as

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per the protocol standardized in the laboratory and instructions provided by the manufacturer. Reverse transcription

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of RNA and PCR analysis were carried out as per the protocol described earlier (Sharma et al., 1999) In brief total

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RNA (2µg) was reverse transcribed using Oligo primers and superscript reverse transcriptase. The cDNA was subjected to 30 cycles of PCR using different forward and reverse primers of pro-inflammatory cytokines,

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inflammatory enzymes and AP-1 factors using appropriate annealing temperatures as indicated earlier (Patil et al., 2015) in a gradient Eppendorf thermocycler. Amplified PCR products were analyzed on 1 % agarose gel using 1X

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TAE buffer. Relative mRNA levels were quantified using image analysis software (ImageJ). The expression of β-

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actin mRNA was used as a positive control and for normalization.

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2.5. Statistical analysis

The experimental data shown as mean ± standard deviation from at least three independent experiments. Statistical

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analysis was done by one-way ANOVA followed by Post hoc Tukey test, Values were considered statistically

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significant if P<0.05, P<0.005.

ACCEPTED MANUSCRIPT 3. Results 3.1. Higher concentration of dexamethasone decreases the viability of A549 cells To study the cytotoxic effect of dexamethasone, A549 cells were treated with and without different concentrations of dexamethasone (0.5-100 µM) for 24 h and the cell viability was determined by MTT assay. Results show that the

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dexamethasone up to 1 µM concentration has no effect on cell viability. While there was a dose dependent decrease

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in cell viability at and above 10 µM concentration of dexamethasone compared to control (Fig. 1). Similar results

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were obtained by counting the viable cells using trypan blue dye exclusion method (data not shown) in a Neubauer

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counting chamber.

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Concentration of dexamethasone (μM)

Fig. 1 Effect of dexamethasone on viability of A549 cells

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Cells were treated with or without different concentrations of dexamethasone in a 96-well plate for 24 h, and the cell viability was measured by MTT assay. Results were expressed as % viability of cells compared to control (mean ± SD, n=8). Values are significantly different from control if, **P<0.005 by student’s t test and one-way ANOVA followed by Post-hoc Tukey test. The results shown here as a representative of three independent experiments

ACCEPTED MANUSCRIPT 3.2a. Dexamethasone inhibits the LPS induced nitric oxide production To assess the effect of dexamethasone on nitric oxide (NO) production, the cells were treated with or without PMA or LPS or dexamethasone alone or LPS with dexamethasone and NO production were measured by Griess reagent. Results show that cells treated with PMA have no effect on nitric oxide production, whereas LPS treatment induces significantly the nitric oxide production by more than 25 % compared to control (Fig. 2). While, treatment of cells

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with dexamethasone at both (0.1 and 1 µM) concentration shows dose dependent decrease in LPS induced nitric

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Nitrite (μM)

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oxide production. Dexamethasone alone (1 µM) also inhibited NO production (17%) compared to control (Fig. 2a).

Fig. 2a Effect of dexamethasone on nitric oxide production in A549 cells.

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A549 cells were treated with LPS (1 μg/ml) or PMA (10 nM) alone or LPS with dexamethasone (DEX) (0.1 or 1 µM) or dexamethasone (1 µM) alone for 24 h and the concentration of nitrite in the spent medium was determined by Griess reagent using sodium nitrite as standard. Data presented as mean ± SD (n=3). Values are significantly different from the control if *P < 0.05 compared with control,

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using one-way ANOVA followed by Post hoc Tukey test. The results shown as representative of three independent experiments

ACCEPTED MANUSCRIPT 3.2b. Dexamethasone inhibits the LPS induced iNOS mRNA expression. Further to confirm that the inhibition of LPS-induced NO production by dexamethasone was due to decreased expression of iNOS, the cells were treated with or without LPS or LPS with dexamethasone, and iNOS mRNA level was analyzed by semi quantitative RT-PCR. Results show that the treatment of cells with LPS significantly induced

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the iNOS mRNA expression by 2 fold compared to control (Fig. 2b). Whereas, cells treated with dexamethasone at

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both (0.1 and 1 µM) concentration dose dependently decrease in LPS induced iNOS mRNA level (Fig. 2b).

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Fig. 2b Effect of dexamethasone on LPS induced iNOS mRNA expression. A549 cells (5×105 cells/well) were treated with or without LPS or LPS with dexamethasone for 24 h. The mRNA levels of amplified genes of RT-PCR were analyzed on 1 % agarose gel, and the band intensity of the experimental samples were compared with the control or LPS alone treatment cells. Data shown are means ± SD from 3 independent experiments. Differences in iNOS mRNA level is statistically significant, if *P < 0.05 compared with controls, #P < 0.05 compared with LPS stimulated values using 1-way ANOVA followed by Post hoc Tukey test. The bar graph represents the densitometric analysis of iNOS mRNA levels

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3.3. Dexamethasone inhibits the PMA induced COX-2 mRNA levels To assess the anti-inflammatory effect of dexamethasone the mRNA level of major pro-inflammatory enzyme COX2 was analyzed by semi quantitative RT-PCR. Results show that the treatment of cells with PMA induced the COX2 mRNA expression by more than 3 fold compared to control (Fig. 3).Whereas, cells treated with dexamethasone at

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1µM concentration decreased the PMA induced mRNA level of COX-2. Cells treated with dexamethasone alone

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also show more than 65% decrease in the expression of COX-2 mRNA levels compared to control (Fig. 3).

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Fig. 3 Effect of dexamethasone on PMA induced COX-2 mRNA expression. A549 cells (5×105 cells/well) were treated with or without PMA or PMA with dexamethasone or dexamethasone alone for 24 h. The mRNA levels of amplified genes of RT-PCR were analyzed on 1 % agarose gel, and the band intensity of the experimental samples were compared with the control or PMA alone treatment cells. Data shown

ACCEPTED MANUSCRIPT are means ± SD from 3 independent experiments. Differences in COX-2 mRNA level is statistically significant, if *P < 0.05 compared with controls, #P < 0.05 compared with PMA stimulated values using 1-way ANOVA followed by Post hoc Tukey test. The bar graph represents the densitometric analysis of COX-2 mRNA levels 3.4. Dexamethasone significantly inhibits the PMA induced mRNAs expression of pro-inflammatory cytokines

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To analyze the effect of dexamethasone on PMA induced expression of pro-inflammatory cytokines, the A549 cells

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were treated with PMA or dexamethasone alone or PMA with dexamethasone for 24h and the total RNA was subjected to RT-PCR. Results show that compared to control the treatment of A549 cells with PMA significantly

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induced the expression of pro-inflammatory cytokines (IL-1β, IL-2, IL-6, IL-8 and TNF-α). Among the proinflammatory cytokines studied at 24 h, maximum induction of more than 3 fold was observed for IL-1β, IL-2, IL-8

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and TNF-α mRNA levels (Fig. 4). While, dexamethasone (at 0.1 µM or 1 µM concentration) treatment significantly

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inhibited the PMA induced mRNA levels of all the above pro-inflammatory cytokines in a dose dependent manner (Fig. 4). Cells treated with dexamethasone (at 0.1 or 1 µM concentration) alone have no effect on IL-2 and IL-8,

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while significantly decreased the expression of IL-1β, IL-6 and TNF-α mRNA levels compared to control (Fig. 4).

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These results suggested that dexamethasone show anti-inflammatory activity in lung cells.

ACCEPTED MANUSCRIPT Control PMA PMA+Dex 0.1 PMA+Dex 1 Dex 0.1 Dex 1

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Fig. 4 Effect of dexamethasone on PMA induced mRNAs expression of pro-inflammatory cytokines in A549

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cells

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A549 cells (5×105 cells/well) were treated with or without PMA or PMA with dexamethasone or dexamethasone alone for 24 h in 6-wells plate. The mRNA levels of amplified genes of semi-quantitative RT-PCR samples were

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analyzed on 1 % agarose gel and the band intensity of the experimental samples were compared with the control and PMA treatment alone. β-actin was used as a positive control and for normalization. Data shown are mean ± SD

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from 3 independent experiments. Differences in proinflammatory cytokines mRNA levels are statistically significant, if *P < 0.05 compared with controls, #P < 0.05 compared with PMA stimulated values using 1-way ANOVA

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followed by Post hoc Tukey test. The bar graph represents the densitometric analysis of mRNA levels. 3.5. Dexamethasone inhibits the LPS induced mRNAs expression of pro-inflammatory cytokines. Further to analyze the effect of dexamethasone on LPS induced mRNAs expression of pro-inflammatory cytokines, A549 cells were treated with LPS or LPS with dexamethasone and the mRNA levels were analyzed by semi quantitative RT-PCR. Treatment of A549 cells with LPS significantly induced the expression of pro-inflammatory cytokines (IL-1β, IL-2, IL-6, IL-8 and TNF-α). Among the pro-inflammatory cytokines studied, maximum induction of more than 2 fold was observed for TNF-α mRNA level (Fig. 5). While, treatment of cells with dexamethasone (at

ACCEPTED MANUSCRIPT 0.1 µM or 1 µM concentration) significantly inhibited the LPS induced mRNA levels of the above pro-inflammatory

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cytokines in a dose dependent manner (Fig. 5).

Fig. 5 Effect of dexamethasone on LPS induced mRNAs expression of pro-inflammatory cytokines in A549 cells Cells were treated with or without LPS (1 μg/ml) or LPS with dexamethasone (0.1 or 1µΜ) for 24 h in 6-wells plate. The mRNA levels of pro-inflammatory cytokines were analyzed by semi-quantitative RT-PCR. Data shown are mean ± SD from 3 independent experiments. Differences in pro-inflammatory cytokine mRNA levels are statistically

ACCEPTED MANUSCRIPT significant, if *P < 0.05 compared with controls, #P < 0.05 compared with PMA stimulated values using 1-way ANOVA followed by Post hoc Tukey test. The bar graph represents the densitometric analysis of mRNA levels. 3.6. Dexamethasone significantly inhibits the PMA induced mRNAs expression of c-Jun, Jun-D and c-Fos AP1 factors To analyze the effect of dexamethasone on the expression of mRNA levels of different AP-1 factors in A549 cells,

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the cells were treated with or without PMA or PMA with dexamethasone or dexamethasone alone for 24h and their

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total RNA was subjected to RT-PCR. Results show that in A549 cells, except Fos-B the Jun (c-Jun, Jun-B, and Jun-

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D) and Fos (c-Fos, Fra-1, Fra-2) family member mRNA transcripts were expressed at different levels. Compared to control the cells treated with PMA induced the expression of c-Jun significantly by 1.5 fold, c-Fos by 2.3 folds and

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Fra-1 by 1.7 fold (Fig. 6). However, PMA marginally increased the expression of Jun-D and Fra-2 mRNA transcripts by 24 h (Fig. 6). Whereas the treatment of cells with dexamethasone (at 0.1µM or 1µM concentration)

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significantly decreased the PMA induced mRNA levels of c-Jun and c-Fos in a dose dependent manner while, marginal decrease was observed with Jun-D mRNA level. No significant inhibition of PMA induced Fra-1 mRNA

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level at 0.1μM but marginal inhibition was observed at 1μM concentration of dexamethasone. Cells treated with dexamethasone (at 0.1µM or 1µM concentration) alone showed marginal decrease in mRNA levels of c-Jun and

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Jun-B compared to control (Fig. 6). These results suggested that c-Jun and c-Fos play a major role in inflammation.

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Fig.6 Effect of dexamethasone on the PMA induced mRNAs expression of AP-1 factors in A549 cells

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Cells were treated with or without PMA or PMA with dexamethasone or dexamethasone alone for 24 h in a 6-wells plate. Total RNA was isolated from control and treated cells. cDNA was prepared by RT and subjected to 30 cycles

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of PCR using specific primers of Jun and Fos family members. Expression of β-actin was used as a positive control and for normalization. Data shown are mean ± SD from 3 independent experiments. Differences in AP-1 factors

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mRNA levels are statistically significant: if *P < 0.05 compared with control, #P < 0.05 compared with PMA

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stimulated values using one-way ANOVA followed by Post hoc Tukey test. The bar graph represents the densitometric analysis of mRNA levels.

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3.7. Dexamethasone significantly inhibits the LPS induced mRNAs expression of c-Jun, Jun-B and c-Fos AP-1 factors.

To analyze the effect of dexamethasone on the expression of mRNA levels of different AP-1 factors in A549 cells, the cells were treated with or without LPS or LPS with dexamethasone for 24h and their total RNA was subjected to RT-PCR. Results show that compared to control the cells treated with LPS induced the expression of c-Jun significantly by 2 fold and c-Fos by 1.5 fold (Fig. 7). However, LPS marginally increased the expression of Jun-B mRNA transcripts. Whereas, the treatment of cells with dexamethasone (at 0.1µM or 1µM concentration)

ACCEPTED MANUSCRIPT significantly decreased the LPS induced mRNA levels of c-Jun and c-Fos, marginal decrease in the mRNA levels of

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Jun-B (Fig. 7) suggested that c-Jun and c-Fos play a major role in inflammation.

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Fig. 7 Effect of dexamethasone on the LPS induced mRNAs expression of AP-1 factors in A549 cells

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Cells were treated with or without LPS or LPS with dexamethasone for 24 h in a 6-wells plate. Total RNA was isolated from control and treated cells. cDNA was prepared by RT and subjected to 30 cycles of PCR using specific

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primers of Jun and Fos family members. Expression of β-actin was used as a positive control and for normalization. Data shown are mean ± SD from 3 independent experiments. Differences in AP-1 factors mRNA levels are statistically significant: if *P < 0.05 compared with control, #P < 0.05 compared with LPS stimulated values using one-way ANOVA followed by Post hoc Tukey test. The bar graph represents the densitometric analysis of mRNA levels.

ACCEPTED MANUSCRIPT 4. Discussion Control of chronic inflammation is important in several inflammatory conditions like asthma, rheumatoid arthritis and fibrosis in lung diseases. The regulatory pathways that control chronic inflammation are complex and multifactorial. Over expression of inflammatory mediators such as inflammatory cytokines, matrix metalloproteinase’s (MMPs) and cyclo-oxygenase (COX-2) reflects chronic inflammation pathology. Therefore these inflammatory mediators are important potential targets for therapeutic intervention (Sakane et al., 1997). Dexamethasone (DEX) is

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a potent long-lasting synthetic glucocorticoid known to inhibit the inflammatory cascade. Because of its potent anti-

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inflammatory effects, it is widely used to treat a variety of acute and chronic inflammatory diseases including acute

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lung inflammation, asthma, and rheumatoid arthritis (Kumar et al., 2003; Goulding, 2004). Glucocorticoids regulate many biological processes through their intracellular glucocorticoid receptors. Following glucocorticoid diffusion

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through the cell membrane bind to its receptors and the activated hormone and glucocorticoid receptor complex translocated into the nucleus and repress transcription factors responsible for the expression of inflammatory

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mediators (Stahn et al., 2007). Hence understanding the mode of action of glucocorticoids plays a central role in the treatment and development of drugs.

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Dexamethasone a potent anti-inflammatory and immunosuppressive glucocorticoid is widely used in the treatment

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of chronic inflammatory diseases (Barnes, 2006; Hillier, 2007). Studies have shown that dexamethasone inhibits the expression of inflammatory mediators in animal models of acute neural injury indicates the action on central

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nervous system (CNS) efficacy (Kurkowska-Jastrzebska et al., 2004; Zhang et al., 2007). Several in vitro studies show that the cytokine induced expression of eotaxin, IL-6, IL-8, GM-CSF, and RANTES were decreased by

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glucocorticoids (Levine et al., 1993; Kwon et al., 1995; Stellato et al., 1995). Hence in the present study, we analyzed the effect of dexamethasone on inflammatory mediators and AP-1 factors in PMA or LPS stimulated lung

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epithelial cells. It is a well-known fact; during bacterial infection the epithelial cells present at the mucosal surface are capable of secreting chemo attractants and pro-inflammatory cytokines, the important mediators in lung inflammation. Hence the lung epithelial cells acts as an important site for glucocorticoid action in asthma and other lung inflammatory diseases (Devalia and Davies, 1993). In our study dexamethasone up to 1 µM concentration show no effect on the cell viability, suggested that the steroids at low concentrations does not exhibit cytotoxic effect in lung cells.

ACCEPTED MANUSCRIPT Nitric oxide (NO) is a short-lived, readily diffusible molecule of great biological importance. It plays a key role in signal transduction, neurotransmission, and host-defense mechanism. NO is produced by nitric-oxide synthases (Alderton et al., 2001; Bermudez et al.) and is highly reactive gaseous mediator involved in many physiologic processes its extensive production lead to various inflammatory diseases (Bogdan, 2001). Hence, the decreased production of nitric oxide is regarded as a therapeutic target for inflammation. In our study A549 cells treated with PMA has no effect on NO production, while LPS induced the NO production. However the treatment of cells with

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dexamethasone significantly decreased the LPS induced NO production in a dose dependent manner suggesting the

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anti-inflammatory activity of dexamethasone. Our results are in good agreement with earlier studies reported by

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Takashi Ozaki et al. who reported that dexamethasone inhibits the NO production in pro-inflammatory cytokinestimulated hepatocytes (Ozaki et al., 2010).

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Inducible nitric oxide synthase (iNOS) is elevated in many inflammatory diseases of the respiratory tract. One of the potential mechanisms by which dexamethasone pretreatment inhibited LPS induced lung inflammation may be a

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decrease in iNOS expression, suggesting that inducible isoform of NOS is responsible for the excess production of NO in LPS induced sepsis in animals (Trifilieff et al., 2000). Studies are reported that in both animal models and

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humans that LPS stimulates iNOS expression and NO over-production, which damages lung tissue through peroxynitrite formation (Razavi et al., 2002). In our present study, the elevated iNOS expressions were detected in

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the cells treated with LPS alone whereas the pretreatment of cells with dexamethasone significantly inhibited the LPS induced mRNA level of iNOS suggesting the inhibitory activity of dexamethasone on LPS-induced NO

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production was also reflected with the suppression of iNOS mRNA expression level as shown by semi-quantitative

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RT-PCR. Similar studies were reported in endotoxin-induced acute lung injury where dexamethasone shows protective effect through inhibiting expression of iNOS (Yu et al., 2009).

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Over expression of the inflammatory cytokines, MMPs and COX-2 act as an important inducer of chronic inflammation. COX-2 catalyzes the biosynthesis of prostaglandins (PGs), and induced expression was observed in cells stimulated with pro-inflammatory cytokines or bacterial lipopolysaccharide (Khanapure et al., 2007; Suleyman et al., 2007). COX-2 is overexpressed in various cancer tissues and this has been linked to inflammation, inflammatory disorders and tumorigenesis (Gilroy et al., 2001; Mantovani et al., 2008). The present study also showed the COX-2 mRNA was expressed at low levels in control A549 cells, but transiently induced by PMA treatment. Whereas, the cells treated with 1 μM concentration of dexamethasone significantly decreased the PMA induced expression of COX-2 mRNA level. Similar observation was made with RAW 264.7 cells and bone

ACCEPTED MANUSCRIPT marrow-derived macrophages (BMMs) where dexamethasone inhibit the LPS induced mRNA expression of COX-2 gene (Joanny et al., 2012). In cancer cells like A549, HeLa, etc., there is always the presence of basal COX-2 expression. As dexamethasone is anti-inflammatory in nature, the addition of dexamethasone alone is marginally reducing the basal COX-2 expression observed in control cells. Many of the inflammatory genes are commonly over-expressed during chronic non-resolving inflammation. Intriguingly, recent data have shown that glucocorticoid administration for 1 h following endotoxin (LPS) challenge is immunosuppressive, administration of the same

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glucocorticoid dose prior to LPS challenge augments immune responses (Frank et al., 2010).

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Glucocorticoids repress transcription of many genes encoding pro-inflammatory cytokines, chemokines, cell

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adhesion molecules and key enzymes involved in the initiation and/or maintenance of the host inflammatory response (Perretti and Ahluwalia, 2000; Smoak and Cidlowski, 2004). Glucocorticoids inhibit the expression of

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inflammatory mediators in macrophages and other cells and have used in the treatment of many immune-mediated inflammatory diseases (Newton, 2000). Our study show that the pretreatment of A549 cells with dexamethasone

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(0.1 or 1 µM) significantly decreased the PMA or LPS induced mRNAs expression of pro-inflammatory cytokines ((IL-1β, IL-2, IL-6, IL-8 and TNF-α) in a dose dependent manner suggested the protective effects of synthetic

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glucocorticoid dexamethasone on LPS-induced inflammation. Our results correlate with the findings of Yamazaki et al who reported that the glucocorticoids inhibit the synthesis of interleukin (IL)-1, TNF-α, IL-1β, IL-6, MMP-I, and

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COX-2 mRNAs expression in SW982 cells (Yamazaki et al., 2003). Human hosts activate multiple TFs such as AP-1 and NF-B that mediate inflammation (Miller et al., 2000; Huang

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et al., 2008). Glucocorticoids play a key role in the suppression of inflammation by inhibiting the transcription of the

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cytokines through binding to the GR and the activated GR interacts with transcription factors, such as AP-1, NF-κB and CCAAT/enhancer-binding protein-β (De Bosscher et al., 2003). AP-1 complexes normally function as positive

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factors in regulating inflammation and the cell cycle. Yet, different combinations of AP-1 members express differential biological effects (Shaulian and Karin, 2002). AP-1 mediates the release of inflammatory mediators such as IL-8 (Yeo et al., 2004) and regulates angiogenesis caused by pathogen infection (Ye et al., 2007). AP-1 also interacts with other TFs to cope up with the pathogen development (Ravichandran et al., 2006) and modulates the expression of inflammatory mediators during infection with Group B Streptococcus (Vallejo et al., 2000) suggested that the biological functions of AP-1 are complex and depends on cell type (Adcock et al., 1994). The ability of GR to repress the activity of NF-B and AP-1 as well as other key immune modulatory transcription factors has been a major focus of research into the mechanisms underlying the (Al-Harbi et al., 2016) anti-inflammatory effects of

ACCEPTED MANUSCRIPT glucocorticoids. Studies reported that dexamethasone inhibits TNF-α induced MCP-1 production via suppression of AP-1 binding activity in human glomerular endothelial cells (Park et al., 2004). A number of mechanisms have been proposed for the anti-inflammatory actions of dexamethasone, including repression of inflammatory cytokine genes by inhibition of transcriptions factors, as well as induction of anti-inflammatory cytokines such as IL-10 (Goulding, 2004). Dexamethasone inhibits LPS-induced Acute Lung Injury through Inhibition of NF-κB, COX-2,

and Pro-inflammatory Mediators in a mouse model (Al-Harbi et al., 2016). Previous studies showed the

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effect of dexamethasone on the expression of c-Jun and c-Fos in different regions of the neonatal brain.

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Dexamethasone-induced shift of the ratio of c-Jun to c-Fos transcript levels in the brainstem of neonatal

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rats towards a predominance of c-Jun may induce the expression of genes that contain AP-1 response elements in the promoters, since the glucocorticoid receptor can be involved in protein–protein

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interactions with the Jun/Jun homodimer of the AP-1 complex (Sukhareva et al., 2016). Hence in the

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present study, we reported the expression pattern of whole set of AP-1 factors (c-Jun, Jun-B, Jun-D and c-Fos, Fra-1 and, Fra-2) except Fos-B in PMA/LPS induced inflammation and also the effect of dexamethasone on it. A549 cells

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treated with PMA induced the expression of c-Jun, Jun-D, Jun-B, Fra-1, Fra-2 and c-Fos at different levels, whereas pretreatment of cells with dexamethasone significantly decreased the PMA induced mRNA levels of c-Jun and c-Fos

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in a dose dependent manner while, Jun-D, Jun-B and Fra-1 were marginally decreased. While in LPS induced inflammation dexamethasone significantly decreases the c-Jun and c-Fos AP-1 factor suggested that the anti-

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inflammatory activity of dexamethasone is through inhibition of AP-1 subunits c-Jun and c-Fos. Our result consistent with the findings of (Adcock et al., 1994) who reported that cytokines induced both c-Jun and c-Fos are

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reduced by dexamethasone in human lung tissue. Our findings suggest that c-Jun and c-Fos are the major AP-1

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factors involved in inflammation and thus AP-1 may be the major transcription factor for the target of classic GRmediated trans-repression. This may be an important molecular mechanism of steroid action in asthma and other chronic inflammatory diseases that need further investigation.

ACCEPTED MANUSCRIPT CONCLUSION: Our study demonstrates that there is a difference in expression of individual AP-1 transcription factors in PMA or LPS stimulated A549 cells. Further dexamethasone inhibits the PMA or LPS induced mRNAs expression of COX-2, Pro-inflammatory cytokines (IL-1β, IL-2, IL-6, IL-8 and TNF-α) and specific AP-1 factors (c-Jun and c-Fos) suggested that c-Jun and c-Fos may play a key role in the anti-inflammatory activity of dexamethasone. Overall, findings from the present study state that the anti-inflammatory activity of dexamethasone is probably based on the

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modulation of downstream transcription factor AP-1 which in turn regulates the expression of pro-inflammatory

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cytokines and inflammatory enzymes. Thus dexamethasone is a possible regulatory molecule(s) in treatment of

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inflammatory diseases.

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ACKNOWLEDGMENTS

The authors wish to express their gratitude to the Department of Microbiology and Biotechnology, Bangalore

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University, Bengaluru, for providing the DST-FIST, UGC-SAP and department facility. Author RHP is grateful to

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UGC-BSR for providing fellowships.

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Funding

This work was supported by the University Grant Commission- Basic Scientific Research (UGC-BSR), University

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Grant Commission-Centre with Potential for Excellence in Particular Area (UGC-CPEPA) [8-2/2008(NS/PE)] and Department of Science and Technology-Promotion of University Research and Scientific Excellence (DST-PURSE)

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[SR/59/Z-23/2010/38(c)], New Delhi.

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Conflict of Interest: Authors declare that there are no conflicts of interest.

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