TLR5 involvement in attenuated IL-8 production in nuclear decorin silenced oral mucosal dysplastic keratinocytes and squamous cell carcinoma

doi:10.4236/ajmb.2013.34032

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American Journal of Molecular Biology, 2013, 3, 248-254 AJMB

http://dx.doi.org/10.4236/ajmb.2013.34032 Published Online October 2013 (http://www.scirp.org/journal/ajmb/)

TLR5 involvement in attenuated IL-8 production in

nuclear decorin silenced oral mucosal dysplastic

keratinocytes and squamous cell carcinoma*

Nyla Dil1,2#, Abhijit G. Banerjee1,3

1Department of Oral Biology, Faculty of Dentistry, University of Manitoba, Winnipeg, Canada

2Department of Immunology, and Department of Medical Microbiology and Infectious Diseases, Faculty of Medicine, University of

Manitoba, Winnipeg, Canada

3Center for Genomic Bio-Medicine and Research Institute, Durg, Chhattisgarh, India

Email: #[email protected]

Received 7 August 2013; revised 3 September 2013; accepted 27 September 2013

License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

Head and neck cancer is one of the most prevalent

cancers in the world. Roughly half of these malignan-

cies originate from oral mucosa and constitute oral

squamous cell carcinomas. Despite many advances in

diagnostic and therapeutic regimens, five-year sur-

vival rate remains at roughly 50%, indicating the

need for in depth understanding of the oral squamous

cell carcinoma immunobiology. We have previously

shown that in human dysplastic oral keratinocytes

(DOK) and malignant squamous cell carcinoma

(SCC-25), multifunctional proteoglycan decorin is

aberrantly expressed and localized in the nucleus

bound to nuclear EGFR. In vitro nuclear decorin

knockdown significantly reduced IL-8 and IL8-de-

pendent migration, invasion and angiogenesis in these

cells. Since toll-like receptor (TLR) signalling leads to

IL-8 production, we examined here if these receptors

played a role in decorin silencing mediated reduction

in IL-8 levels in oral mucosal d ysplast ic keratino cytes

and squamous carcinoma cells. Decorin silenced DOK

and SCC-25 cells showed a marked diminution of

TLR5 mRNA and protein expression compared with

respective controls that translated into the loss of func-

tion in response to appropriate TLR ligand. In these

mucosal oral epithelia , decorin stable kno ckdown sig-

nificantly down-regulated IL-8 production following

activation with TLR5 ligand flagellin. These data sug-

gest that decorin silencing interferes with IL-8 pro-

duction, in part, by altering TLR5 expression and

signaling in dysplastic and malignant oral epithelia.

This study highlights the significance of TLR5 ex-

pression and signaling and its plausible interactions

with proteoglycans in mucosal cancers.

Keywords: Oral Cancer; Mucosal Cancer; Nuclear

Decorin; TLR5; IL-8; Flagellin; PAMP; DAMP; SLRP;

Proteoglycans

1. BACKGROUND

Head and neck squamous cell carcinoma (HNSCC) is a

devastating disease that accounts for roughly 1.6 million

newly diagnosed cases and 330,000 deaths each year

worldwide [1,2]; half of these cancers are localized in the

oral cavity and are termed oral squamous cell carcinoma.

A multifactorial mucosal cancer, oral squamous cell car-

cinoma, is primarily associated with chronic tobacco,

alcohol use and betel chewing; however, chronic in-

flammation, viral infections (human papillomavirus),

genetic predisposition and poor oral hygiene have also

been strongly implicated in oral tumorigenesis [3-5].

The majority of inflammatory conditions are triggered

by Toll-like receptors (TLRs) which are a large family of

at least 11 evolutionarily conserved proteins character-

ized by ligand specific activation and complex down-

stream signaling [6-8]. TLRs are predominantly ex-

pressed on various immune and structural cells where

they serve as innate cell surface pathogen sensors and

promote local inflammation by triggering the induction

of various proinflammatory mediators, including IL-8.

Recently, TLR expression or up-regulation has been de-

tected in various tumour types, especially in epithelium

derived cancers [9-11]. Expression of TLRs varies in

different cancerous cell types; however, evidence indi-

*Competing Interests: No financial or non-financial competing interests

exist.

#Corresponding author.

OPEN ACCESS

N. Dil, A. G. Banerjee / American Journal of Molecular Biology 3 (2013) 248-254 249

cates that TLR expression is functionally associated with

tumorigenesis. It has been suggested that TLR expression

might promote malignant transformation of epithelial

cells [11,12]. Engagement of TLRs promotes tumor de-

velopment and metastasis, protects the cancerous cells

from immune attack and induces resistance to apoptosis

and chemo-resistance in some malignancies [10,12-14].

In addition to its putative proinflammatory functions,

IL-8 is a potent angiogenic factor and plays an important

role in cancer progression and metastasis [15-18]. IL-8 is

responsible for most of the angiogenic activity induced

by human oral mucosal dysplastic and malignant epithe-

lial cells [19,20]. Previously, we have shown that in hu-

man dysplastic oral keratinocytes (DOK) and malignant

squamous carcinoma cells (SCC-25), multifunctional

proteoglycan decorin is atypically expressed and local-

ized in the nucleus bound to nuclear epidermal growth

factor receptor (EGFR) [18,21]. Post-transcriptional nu-

clear decorin knock-down resulted in significantly re-

duced IL-8 and IL8-dependent migration, invasion and

angiogenic potential in these dysplastic and malignant

oral mucosal epithelia [18,20]. Since TLR signaling is an

upstream event in the majority of cytokines/chemokine

expression, in this current study, we examined if TLR

pathway is implicated in the aberrant nuclear decorin

silencing mediated attenuated IL-8 production.

2. MATERIALS AND METHODS

2.1. Cell Lines and Culture Conditions

Oral mucosa origin, premalignant-Dysplastic Oral Kera-

tinocytes (DOK) and malignant-Squamous Carcinoma

(SCC-25) cell lines were routinely maintained in

DMEM/F12 (Hyclone, Logan, Utah) supplemented with

10% Foetal Calf Serum for use as in vitro model in our

studies, as described previously [20,22,23]. Silencing of

decorin gene expression was achieved using short hairpin

RNA (shRNA) technology as described earlier [18].

Briefly, oligonucleotides targeting decorin transcript va-

riants A1 and A2 and scrambled sequence control were

custom synthesized, annealed, and cloned into shRNA

expression vector pGeneClip PuroTM (Promega) by Super

Array Bioscience Corporation (Frederick, MD). BLAST

queries were performed to ensure that the sequences have

no significant homology with any other human genes.

Transformation grade shRNAi plasmids were transfected

into DOK and SCC-25 cells using Effectene™ transfec-

tion reagent following manufacturer’s protocol (Qiagen,

Valencia, CA). The stable transfectants were selected for

puromycin (Calbiochem, San Diego, CA) resistance at

2.5 µg/ml optimal concentration. Pools of stable trans-

fectants (maintained at 1 µg/ml of puromycin) were used

in all experiments to avoid clone-specific differences.

Decorin knock down was confirmed at transcript and

protein level by quantitative real-time reverse transcrip-

tion-PCR and Western blotting, respectively. Pooled

decorin-shRNA transfected DOK or SCC-25 clones

showed more than 80% reduction in decorin mRNA ex-

pression and almost complete abrogation of decorin pro-

tein expression in nuclear lysates and/or in whole cell

lysates when compared to control-shRNA transfected

clones or no transfection wild type DOK [18]. Herein,

untransfected DOK and SCC-25 cells will be referred to

as wild type (WT), scrambled shRNA stable transfectants

as control (or Ctrl-shRNA in figures), and decorin

shRNA stable transfectants as decorin silenced (or DCN-

shRNA in figures).

2.2. Multiplex PCR

The transcript expression levels of innate immune TLRs

and co-regulatory molecules were quantified in decorin

silenced, control, and WT DOK and SCC25 cells using

multiplex PCR (MPCR) kit for human signaling receptor

set-2 (TLR1, TLR2, TLR3, TLR5, TLR6, TLR9 and

CD14) from Maxim Biotech, San Francisco, CA. This

set also included housekeeping gene-GAPDH, as internal

cDNA loading control in each reaction. MPCR was car-

ried out according to the manufacturer’s instructions.

Briefly, 1X MPCR buffer, 2.5 units of Taq DNA poly-

merase, and cDNA template from DOK and SCC25 cells

were mixed in a 25 µl reaction and subjected to 35 cycles

of PCR, with denaturing, annealing, and extension tem-

peratures at 96˚C, 67˚C, and 70˚C, respectively. Follow-

ing MPCR, the DNA amplicons were fractionated elec-

trophoretically on 2% agarose gel containing 0.5 µg/ml

ethidium bromide.

2.3. Real-Time PCR

RNA was extracted from DOK and SCC-25 cells using

RNeasy Plus mini kit (Qiagen, Valencia, CA) and 2.5 g

of total RNA was used to synthesize cDNA, using Su-

prescript III Reverse Transcriptase (Invitrogen, San

Diego, CA). Quantitative RT-PCR was performed using

QuantiTect SYBR Green PCR kit (Qiagen, Valencia,

CA) on the Mini Opticon Real-Time PCR system

(BioRad, Hercules, CA) as per manufacturer’s protocol.

Quantitative PCR primer pairs were designed for SYBR

Green chemistry based detection of amplicons for TLR5

(5’-TGCATTAAGGGGACTAAGCCT-3’,

5’-AAAAGGGAGAACTTTAGGGACT-3’) and

GAPDH (5’-ACAGTCAGCCGCATCTTCTT-3’,

5’-GTTAAAAGCAGCCCT G GTGA-3’). GAPDH was

used as relative house-keeping gene expression control to

normalize for sample variations.

2.4. Western Blot Analysis

Cells were rinsed with ice-cold PBS and were lysed in a

N. Dil, A. G. Banerjee / American Journal of Molecular Biology 3 (2013) 248-254

250

buffer containing 20 mM Tris, pH 7.6, 0.1% SDS, 1%

Triton-X, 1% deoxycholate, 100 µg/ml PMSF, and pro-

tease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO).

Lysates were centrifuged at 20,000 x g for 20 min at 4˚C.

Protein concentration was determined by Bis-Cinchonic

Acid (BCA) protein assay (Pierce, Rockford, IL) and

subjected to 10% SDS-PAGE analysis, followed by

transfer to polyvinylidene difluoride membrane (Bio-Rad,

Hercules, CA). The membranes were immunoprobed

with 1:500 dilution of monoclonal antibody to human

TLR5 (Alexis Biochemicals, San Diego, CA) or 1:1000

dilution of anti-human beta-tubulin polyclonal antibody.

Western blots were developed with appropriate horserad-

ish peroxidase conjugated secondary antibodies (Bio-Rad)

and ECL Plus chemiluminescence system (Amersham,

Arlington Heights, IL) and exposed to auto radiographic

films. Radiographs were scanned and densitometry

analysis was done using AlphaEase FC software (Alpha

Innotech Corporation, San Leandro, CA).

2.5. ELISA for IL-8 Quantification

Decorin silenced, control and WT DOK and SCC-25

cells (5 × 105 cells/well) were cultured in complete me-

dium in 24-well flat-bottom plates at a final volume of a

500 µl. Cells were stimulated with varying concentra-

tions of flagellin (Alexis Biochemicals, San Diego, CA);

100 ng/ml concentration was found to be optimal. Cul-

ture supernatants were collected after 24 h, 48 h or 72 h

of incubation and IL-8 was assayed with 100 μl of cell

free culture supernatant using DuoSet IL-8 ELISA kit (R

& D Systems, Minneapolis, MN) according to manufac-

turer’s instructions. Absorbance was read at 450 nm with

the SPECTRAMax 190 microplate spectrophotometer

and results were analyzed by SOFTMax Pro software

(Molecular Devices, Sunnyvale, CA). Sample concentra-

tions were determined by interpolation from the standard

curve. IL-8 lower detection limit was found to be 5.6

pg/ml. Samples were read in triplicate.

2.6. Statistical Analysis

Student’s paired t test was used to determine the statisti-

cal significance of the data. Statistical analysis was per-

formed on Graph Pad Prism Software. Significance was

evaluated at p values:

*p < 0.05, **p < 0.01, ***p < 0.001.

3. RESULTS

3.1. TLR5 Expression down Regulation in

Decorin Silenced DOK and SCC25 Cells

Toll-like receptor expression has been described in many

cancers especially epithelial derived tumours and has

been linked to tumour progression [24]. Many of the

TLRs are known to trigger IL-8 expression. We sought to

determine whether nuclear decorin silencing has an ef-

fect on any or all of the TLRs expression in dysplastic

and malignant oral epithelial cells. Multiplex PCR analy-

sis showed that out of an array of TLRs, TLR5 expres-

sion was significantly reduced in decorin silenced DOK

and SCC-25 cells compared to respective WT and con-

trol cells (Figure 1). Interestingly, no difference was

observed in the expression of TLR1, TLR2, TLR3, TLR6,

TLR9 and CD14 between decorin silenced and WT/con-

trol DOK or SCC-25 cells (Figure 1). We confirmed this

observation by Real time PCR analysis using specific

primers; no difference was observed in the expression of

TLR1, TLR2, TLR3, TLR4, TLR6, TLR8 and TLR9

between decorin silenced and WT or controls dysplastic

or malignant epithelial cells (data not shown). However,

Real time PCR analysis using TLR5 specific primers

showed more than 75% reduction in TLR5 expression in

decorin silenced DOK and SCC-25 cells (Figure 2(a)).

To investigate if this reduction in TLR5 expression

translates into lower TLR5 receptor levels, we performed

Western blot analysis and observed similar TLR5 protein

reduction in decorin silenced DOK and SCC-25 cells in

comparison to TLR5 protein expression in respective WT

and/or control cells (F igures 2(b) and (c)).

3.2. Attenuated Flagellin-Induced IL-8

Production in Decorin Silenced DOK

and SCC25 Cells

We have shown previously that nuclear decorin silencing

results in attenuated constitutive IL-8 production in oral

mucosal DOK and SCC-25 cells. Here, we sought to

determine if nuclear decorin silencing-mediated TLR5

down regulation has an effect on IL-8 induction in these

dysplastic and malignant oral epithelial cells. Flagellin is

a known ligand for TLR5 and flagellin stimulation of

Figure 1. Toll-like receptors and CD14 expression in decorin

silenced and WT dysplastic and malignant oral epithelial cells.

DOK and SCC-25 cells were stably transfected with decorin-

shRNA (DCN-shRNA), or scrambled sequence-shRNA (Ctrl-

shRNA) or no transfection control (WT). RNA was extracted

and cDNA was subjected to multiplex RT-PCR to detect mul-

tiple pattern recognition receptor transcripts. MPCR products

were fractionated electrophoretically on a 2% agarose gel con-

taining 0.5mg/ml ethidium bromide, visualized under UV

light and photographed. The results shown are representative

of three independent experiments.

N. Dil, A. G. Banerjee / American Journal of Molecular Biology 3 (2013) 248-254 251

(a)

(b)

(c)

Figure 2. TLR5 down regulation in decorin silenced DOK

and SCC25 cells. RNA was extracted from WT, control and

decorin silenced DOK and SCC-25 cells and (a) cDNA was

subjected to quantitative RT-PCR, normalized TLR5 expres-

sion from one representative experiment of three. (b) Cell

lysates were collected as described in materials and methods

and subjected to SDS-PAGE followed by immunoblotting

using anti-TLR5 and antitubulin antibodies. (c) Densitomet-

ric analysis is presented as a histogram of TLR5 relative

band density from 3 experiments. ***p < 0.001 compared to

respective controls.

epithelial cells results in increased IL-8 production. To

investigate the TLR5 link in IL-8 mitigation in decorin

silenced dysplastic and malignant epithelia, we deter-

mined and compared the levels of IL-8 production upon

flagellin stimulation in these cells. Briefly, cells were

stimulated with flagellin for 24, 48 and 72 h; 24 h time

point was considered optimal for IL-8 production. Con-

sistent with down regulation of TLR5 expression levels,

we found a significant reduction in flagellin stimulated

IL-8 production in decorin silenced cells compared to

WT or ctrl-shRNA treated DOK or SCC-25 cells (Figure

3). It is interesting to note that SCC-25 cells produce

much higher levels of flagellin stimulated IL-8 compared

to dysplastic oral keratinocytes.

4. DISCUSSION

A number of functions have been assigned to small leu-

cine-rich proteoglycan (SLRPs) decorin. The most in-

triguing function is the ability to inhibit growth and me-

tastasis of a range of tumors. Accordingly, decorin is

being studied extensively as a naturally occurring poten-

tial oncosuppressive agent [25]. Decorin is normally pre-

sent in the extracellular stromal compartment and analy-

ses of various tumors indicate that it is rarely expressed

by cancer cells. However, studies by us and others indi-

cate that there are exceptions to this prevailing decorin

expression and decorin mediated cancer growth and sup-

pression model [21,26,27]. Ectopic decorin expression

has been linked to increased vascular endothelial growth

factor expression and increased angiogenesis [28]. In our

previous studies of oral precancerous and cancerous le-

sions and cellular models of oral cancer progression, we

demonstrated that decorin is aberrantly expressed and

localized in the nucleus bound to nuclear epidermal

growth factor receptor (EGFR) in the dysplastic and ma-

Figure 3. Reduced Flagellin-induced IL-8 production in

decorin Silenced DOK and SCC25. Cells were cultured with

100 ng/ml flagellin and IL-8 was measured in 24 h culture

supernatants using ELISA. Data are presented as mean ± SD

of three replicates of one representative experiment of four.

***p < 0.001 compared to respective controls.

N. Dil, A. G. Banerjee / American Journal of Molecular Biology 3 (2013) 248-254

252

lignant oral epithelial cells [18,21]. Stable knock down of

nuclear decorin resulted in significant down regulation of

IL-8 and IL-8 dependent migration, invasion and angio-

genic potential in this oral cancer cell line model. Here,

we show that reduced IL-8 in nuclear decorin silenced

DOK and SCC-25 cells is mediated, in part, by reduced

TLR5 expression and signaling.

Toll-like receptors are a major component of an evolu-

tionarily conserved, classic pattern recognition system,

which underpins pathogen recognition, mediates inflam-

matory responses and bridges innate and adaptive immu-

nity. Historically, TLR agonists have been used as adju-

vant in anti cancer immunotherapy. However, a growing

body of clinical and experimental studies suggest that the

neoplastic process may actually subvert TLR signalling

pathway to enhance cancer progression. Hence, the TLR

system appears to play the role of a double-edged sword

in the neoplastic development.

Increased expression of various TLRs has been found

to be associated with a variety of cancers of gastrointes-

tinal, oral and genital mucosa [11,29]. Of all the TLRs,

TLR5 is the most prominent TLR that appears to have a

role in mucosal neoplastic processes. Enhanced TLR5

expression has been reported in gastric and colorectal

malignancies, as well as in precancerous lesions of uter-

ine cervix [30,31]. Stimulation with TLR5-ligand, flagel-

lin, increased proliferation and induced TLR5 mediated

IL-8 production in various gastric cancer cell lines [32].

A role for TLR5 has also been described in mucosal head

and neck cancers. In biopsy sample of 119 oral tongue

squamous cell carcinoma patients, TLR5 expression was

found to be more abundant and widespread in cancer

cells than in the adjacent healthy lingual epithelial tissue

[33]. In this oral cancer cohort, TLR5 expression levels

predicted prognosis in terms of recurrence and patient

survival. In salivary gland adenocarcinoma, flagellin ac-

tivation of TLR5 promoted migration and invasion of

cancer cells [12]. Hence, increased TLR5 expression is

correlated with mucosal cancer progression and, there-

fore, has been proposed as a biomarker for gastric and

cervical dysplasia as well as oral cancer and might help

identify dysplastic lesions in mucosal epithelium [30,31,

33].

The canonical function of proteoglycans is the main-

tenance and regulation of the architecture of various ex-

tracellular matrices (ECM). Decorin is an important

structural component of the ECM and a fundamental

regulator of collagen fibrillogenesis that got its eponym

based on its ability to decorate collagen fibrils. However,

a growing body of recent evidence indicates that secreted

proteoglycans in particular those from the small leucine-

rich family (including decorin), hyaluronan-binding gene

family as well as the glycosaminoglycan hyaluronan act

as endogenous ligand for TLR2, TLR4 and TLR6 in ad-

dition to their above mentioned widely recognized struc-

tural function [34,35]. These proteoglycans and other

endogenous molecules act as “danger signals” and can

activate pattern recognition receptors and thus are re-

ferred to as damage-associated molecular patterns

(DAMPs) [36]. Any direct or indirect interaction be-

tween decorin and TLR5 has hitherto not been described.

We show here for the first time that under-expressing

aberrant nuclear localized decorin results in down regu-

lation of TLR5 and TLR5 mediated IL-8 production in

dysplastic and malignant oral epithelial cells. This might

be due to nuclear decorin interactions with its binding

partner nuclear EGFR (which acts as a transcription fac-

tor) or additional interactions with other nuclear and/or

cytosolic factors. Further studies are warranted to deci-

pher these interactions and to explore how exactly the

gene expression of TLR5 is regulated by nuclear local-

ization of decorin in these cells.

5. CONCLUSION

Using shRNA expression plasmid mediated in vitro sta-

ble nuclear decorin silencing, we present here a study of

the involvement of TLRs in attenuated IL-8 production

upon nuclear decorin knockdown in dysplastic and ma-

lignant oral mucosal epithelial cells. We demonstrate that

nuclear decorin knockdown significantly inhibits TLR5

(a putative mucosal cancer biomarker) and TLR5 medi-

ated IL-8 production in these cells. These findings and

our previous work on nuclear decorin suggest that nu-

clear decorin silencing suppresses major tumor devel-

opment and progression processes such as IL-8 depend-

ent migration, invasion, and angiogenesis in part by miti-

gating the expression of TLR5. This study also highlights

the significance of TLR5 expression and signaling in de-

velopment and progression of mucosal cancers. It might

be potentially useful in developing a therapeutic regimen

for patients presenting with premalignant or malignant

oral mucosal lesion accompanied with poor oral health

associated mucosal inflammation.

6. AUTHORS’ CONTRIBUTIONS

ND contributed to study concept, performed, and ana-

lyzed the experiments, interpreted data and wrote the

manuscript. AGB conceptualized nuclear decorin study

and designed and BLAST decorin targeting oligonucleo-

tides, analyzed results and interpreted data, and approved

the final manuscript and directed the research program.

All authors reviewed and approved the final manuscript.

7. ACKNOWLEDGEMENTS

The authors gratefully acknowledge Dr. J.K. Vishwanatha, University

of North Texas, Fort Worth for providing the oral cancer progression

model cell lines and Linda Delmage, Charlton Cooper, Susan Pylypas,

N. Dil, A. G. Banerjee / American Journal of Molecular Biology 3 (2013) 248-254 253

University of Manitoba for technical assistance. This project was sup-

ported by research grants form Manitoba Health Research Council and

Manitoba Medical Service Foundation and by funds from Department

of Oral Biology and Faculty of Dentistry, University of Manitoba.

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