- Research article
- Open Access
DNA demethylation-dependent enhancement of toll-like receptor-2 gene expression in cystic fibrosis epithelial cells involves SP1-activated transcription
© Furuta et al; licensee BioMed Central Ltd. 2008
- Received: 17 December 2007
- Accepted: 21 April 2008
- Published: 21 April 2008
The clinical course of cystic fibrosis (CF) is characterized by recurrent pulmonary infections and chronic inflammation. We have recently shown that decreased methylation of the toll-like receptor-2 (TLR2) promoter leads to an apparent CF-related up-regulation of TLR2. This up-regulation could be responsible, in part, for the CF-associated enhanced proinflammatory responses to various bacterial products in epithelial cells. However, the molecular mechanisms underlying DNA hypomethylation-dependent enhancement of TLR2 expression in CF cells remain unknown.
The present study indicates that there is a specific CpG region (CpG#18-20), adjacent to the SP1 binding site that is significantly hypomethylated in several CF epithelial cell lines. These CpGs encompass a minimal promoter region required for basal TLR2 expression, and suggests that CpG#18-20 methylation regulates TLR2 expression in epithelial cells. Furthermore, reporter gene analysis indicated that the SP1 binding site is involved in the methylation-dependent regulation of the TLR2 promoter. Inhibition of SP1 with mithramycin A decreased TLR2 expression in both CF and 5-azacytidine-treated non-CF epithelial cells. Moreover, even though SP1 binding was not affected by CpG methylation, SP1-dependent transcription was abolished by CpG methylation.
This report implicates SP1 as a critical component of DNA demethylation-dependent up-regulation of TLR2 expression in CF epithelial cells.
- Cystic Fibrosis
- TLR2 Expression
- Caffeic Acid Phenethyl Ester
- Cystic Fibrosis Airway
- Caffeic Acid Phenethyl Ester
The innate immune system recognizes conserved microbial products, termed pathogen-associated molecular patterns (PAMPs), that are invariant among diverse groups of microorganisms. PAMPs are recognized by a set of germ-line encoded pattern recognition receptors (PRRs) including toll-like receptors (TLRs) [1–3]. After the recognition of microbial PAMPs by innate immune systems, TLRs activate signaling pathways that induce inflammatory cytokines and antimicrobial peptides to eliminate invading pathogens. Eleven members of the mammalian TLR family have been identified and cloned thus far. TLR2 and TLR4 have been studied in the greatest depth [4, 5], and TLR4 appears to be primarily involved in the recognition of lipopolysaccharide (LPS) from Gram-negative bacteria . In contrast, TLR2 responds to a variety of both Gram-negative and Gram-positive bacterial products, including peptidoglycan, lipoprotein, lipoteichoic acid, and lipoarabinomannan. This suggests that TLR2 plays a critical role in the host defence system .
TLR2 is primarily expressed in monocytes, macrophages, dendritic cells, and granulocytes. There is also a growing body of evidence indicating that TLR2 is inducibly expressed in epithelial tissues. Previous studies have shown that TLR2 is expressed at a low level in human epithelial cells under physiological conditions. However, TLR2 expression is greatly increased during bacterial infections through an NF-κB-dependent mechanism [6, 7]. Inducible expression of human TLR2 by the proinflammatory cytokine, TNFα, was also indicated in lung Type II-like A549 epithelial cells . These findings indicate that increased TLR2 expression during bacterial infection may contribute in accelerating the immune response to invading pathogens.
Although optimal TLR2 signaling is required to activate epithelial cells against microorganisms, excessive or inappropriate TLR2 expression and signaling could contribute to hyperresponsiveness against bacterial ligands, and enhance the inflammatory responses frequently detrimental to the host [6, 9–12]. Our previous studies indicate that expression of TLR2 is higher in the airway mucosae of chronic otitis media patients than in normal subjects , and is also associated with inflammatory bowel disease (IBD) such as Crohn's disease [11, 12]. These reports implicate hyperresponsiveness to bacterial infection as a cause of the enhanced susceptibility to chronic inflammation. Thus, regulation of TLR2 expression is likely an important immune-regulatory mechanism, commonly involved in host defence against bacterial infections.
The clinical course of cystic fibrosis (CF), the most common lethal inherited disorder in Caucasians, is characterized by two major respiratory symptoms, recurrent pulmonary infection and chronic inflammation that ultimately lead to death from respiratory failure [13, 14]. Two previous studies [15, 16] showed a modest up-regulation of TLR2 in CF airway epithelial cells that is consistent with an increased proinflammatory response to TLR2-activating bacterial ligands found in CF airways. In accordance with these findings, an increase in the expression of TLR2 gene in CF epithelial cells due to the hypomethylation of the TLR2 promoter has been demonstrated . However, the molecular mechanisms underlying this hypomethylation-dependent activation of TLR2 transcription in human CF epithelial cells remain unknown.
The present study shows that a specific CpG region, adjacent to an SP1 binding site, are hypomethylated in CF epithelial cells. The minimal region required for maintenance of basal TLR2 promoter activity was comprised of the CpG site adjacent to an SP1 binding site. Furthermore, SP1-dependent transcriptional activity, but not SP1 binding, was shown to be elevated in the hypomethylation-dependent enhancement of TLR2 gene expression in CF epithelial cells.
Identification of CF-specific methylation patterns within the human TLR2 promoter
Oligonucleotides used for this study
Primers used for the generation of TLR2 promoter/reporter constructs
Primers used for the generation of mutant TLR2 promoter/reporter constructs a
Primers used for semi-quantitative RT-PCR
5'- CATGTATTCCATCACCACCAG -3'
Oligonucleotides used for EMSA b
5'-GTGCCCCGTGGAAGG TTA GG TTCCCGCACCCCAG-3'
5'-CTGGGGTGCGGGAACC TAA CC TTCCACGGGGCAC-3'
Methylation of CpG sites in the TLR2 promoter is significantly different between non-CF and CF epithelial cells a
7.277 × 10-5***
Identification of the minimal promoter region required for basal TLR2 promoter activity in human epithelial cells
To further restrict a minimal region required for TLR2 basal promoter activity, we constructed pGL3-T2P (-60) plasmid. This construct contains only one E26 transforming-specific (ETS) site and one SP1 binding site (Fig. 2C). The luciferase activity in the cells transfected with pGL3-T2P (-60) was 3-fold higher than in those transfected with pGL3-T2P (-12), indicating that the minimal promoter is located between -60-bp to -12-bp upstream of the transcription start site (Fig. 2C). It is important to note that this minimal region comprises CpG#18-20, and is significantly hypomethylated in CF epithelial cells. These findings support the notion that this region regulates TLR2 expression.
SP1 binding site adjacent to CpG#18-20 in human epithelial cells
Regulation of TLR2 promoter activity and its gene expression by SP1
Inhibition of basal and SP1-induced TLR2 promoter activity by in vitro DNA methylation
SP1 and TLR2 expression
Methylation of CpG#18-20 and SP1 binding
In mammalian cells, CpG methylation in a promoter is a primary epigenetic mechanism for silencing genes, and is involved in the control of cellular function and homeostasis. It is therefore reasonable to predict that aberrant methylation of CpG regions within a promoter could manifest in a pathology associated with some chronic diseases. Recent studies have shown that TLR2 promoter hypomethylation is associated with increased expression of TLR2 in CF bronchial epithelial cells. This is consistent with CF airway pathology that typically shows increased proinflammatory responses to TLR2 bacterial ligands . The present study shows that a specific CpG region of the TLR2 promoter, adjacent to an SP1 binding site, is significantly demethylated, and that this promoter region is both necessary and sufficient to maintain basal TLR2 promoter activity in human epithelial cells. These data suggest that there are CFSMPs (CF-specific methylation patterns) within TLR2 promoter, that might be used as markers to facilitate the discovery of anti-inflammatory CF drugs, as has been the case with anti-tumor drug discovery [20, 21].
The modulation of SP1 binding to its target sequences by DNA methylation is controversial. In the promoters of p21Cip1, 11-hydroxysteroid dehydrogenase type 2, and GSTP1 (glutathione S-transferase p1), SP1 binding was found to be diminished by DNA methylation [22–24]. In contrast, SP1 binding to the claudin-4 (CLDN4) promoter was not influenced by DNA methylation . It was found that the methyl-CpG-binding domain protein 2 (MBD2) was recruited to CpG sites and silenced the CLDN4 gene in ovarian cancer cell lines without interfering with SP1 binding. The present study clearly indicates that SP1 binding to a TLR2 promoter region was not inhibited by DNA methylation. This is similar to what was observed for the CLDN4 promoter . Recruitment of methyl binding proteins to the SP1 binding site in the TLR2 promoter needs to be verified. MBD2 and/or the methyl-CpG-binding protein 1 (MeCP1) complex, a large protein complex that includes MBD2 , could be candidate "X" factors that interact with the methylated TLR2 promoter.
It is still unclear how the methylation patterns of the TLR2 promoter are determined in non-CF and CF epithelial cell lines. Whether it is recruitment of factors required for transcriptional activation or the recruitment of factors required for silencing requires further study. The A549 and HeLa, cell lines used as non-CF epithelial cells in this study, are not thought to express CFTR mRNA, yet their TLR2 expression was comparable to that of the 16HBE14o- cells. Therefore, it appears that wt CFTR expression may not be the only factor that would determine the TLR2 gene expression in epithelial cells. However, the processing of ΔF508CFTR might affect the transcription of the TLR2 gene through a mechanism that involves the methylation of DNA and the expression of specific transcriptional regulatory factors. Preliminary studies comparing CF and non-CF cells showed no difference in the expressions of DNA methyl transferase genes, DNmt1, DNmt3a, and DNmt3b [27, 28], or in the DNA demethylases, MBD2 and Gadd45a [27, 29], (unpublished data). One possible mechanism could involve the recruitment of factors that releases the methylation-induced transcription block by activating demethylation of the promoter in the nucleus. Clearly, further investigation is required to elucidate these mechnaisms.
There appear to be CF-associated methylation patterns within the human TLR2 (hTLR2) promoter, that lead to increased expression of the TLR2 gene in CF epithelial cells. Moreover, SP1-dependent transcription apppears to be an important component of the molecular mechanisms underlying CFSMPs-related aberrant regulation of TLR2 expression in CF epithelial cells. Increased responsiveness to TLR2 ligands as well as an increase in TLR2 expression in CF epithelial cells have been proposed as a contributing factor in CF-associated chronic inflammation. Therefore, CF-specific methylation patterns within the TLR2 promoter may have important implications for the development of therapies directed at sites regulating TLR2 expression. Furthermore, the studies presented here indicate that CF-specific hypomethylation of the hTLR2 promoter may influence regulation of other inflammation-associated genes that are aberrantly regulated in CF epithelial cells [30–34]. Further study of this system may provide insight into the molecular mechanisms regulating inflammation in CF. This study underscores the role that epigenetic mechanisms like DNA methylation play in the modulation of gene expression in response to cellular insult.
5-azacytidine was purchased from Nacalai Tesque (Japan). Mithramycin A was purchased from Sigma (St. Louis, MO, USA). Caffeic acid phenethyl ester (CAPE) was purchased from Calbiochem (Darmstadt, Germany).
16HBE14o- , CFBE41o-  and CFTE29o-  cells were previously generated and grown in Fibronectin/Vitrogen/BSA-coated flask in MEM (Invitrogen, Carlsbard, CA) . CFPAC-1 cells  were purchased from ATCC and grown in Iscove's modified Dulbecco's medium (IMDM) (Invitrogen). The media were supplemented with 10% fetal bovine serum (FBS), 100 mg/ml of penicillin and 100 U/ml of streptomycin. The CFBE41o-, CFTE29o- and CFPAC-1 cells were derived from CF patients that are homozygous for the ΔF508 CF transmembrane conductance regulator (ΔF508CFTR) mutation. The non-CF epithelial cells in this study, 16HBE14o-, A549 and HeLa, were homozygous for wild type (wt)CFTR. Expression of CFTR mRNA in A549 and HeLa cells has not been confirmed.
Sodium bisulfite DNA Sequencing
Genomic DNA was isolated from various cell types using the Blood and Cell Culture DNA Mini kit (Qiagen, Bothell, WA, USA) according to the manufacturer's protocol. Sodium bisulfite DNA sequencing analysis of the TLR2 promoter has been previously described . Briefly, PCR products were subcloned into the pCR2.1-TOPO vector (Invitrogen, Carlsbad, CA, USA), and > 10 clones from each cell line were sequenced. The frequency of methylated CpG at each CpG site and the average % methylation at each CpG site in the TLR2 proximal promoter were calculated. The amount of methylated and unmethylated DNA at each CpG site was compared in non-CF and CF epithelial cells, and then statistically analyzed using Fischer's exact test.
Full-length human TLR2 (hTLR2) promoter constructs, were generated by amplifying different lengths up to 309-bp 5' of the TLR2 gene coding region using genomic DNA prepared from A549 cells as the template for High Fidelity AccuPrime™ Taq DNA Polymerase (Invitrogen). The PCR primers used in the amplification are indicated in Table 1. The PCR products generated were cloned into pCR2.1-TOPO vector using the TOPO-TA cloning kit (Invitrogen). The TLR2 promoter-luc reporter construct was generated by subcloning the TLR2 promoter fragment into the Xho I-Hind III sites of the pGL3-Basic vector (Promega, Madison, WI, USA). Mutant TLR2 promoter constructs were generated utilizing the QuikChange II site-directed mutagenesis kit from Stratagene (La Jolla, CA, USA) according to the manufacturer's instructions. The oligonucleotide primers used to generate the mutants are shown in Table 1. Mutant constructs were generated using either the pGL3-T2P(-120) or the pGL3-T2P(-60) construct as template. The pGL3-T2P(-120) plasmid was either methylated or mock-methylated in the presence or absence, respectively, of Sss I methylase. Construct methylation was confirmed by digestion with methylation-dependent Hap II and methylation-independent Msp I enzymes. The TLR2 expression plasmid and the luciferase reporter construct, IL-8-luc, were described previously . The NF-κB-luc construct was purchased from Stratagene (La Jolla, CA, USA). Expression plasmids for SP1 and SP3 were kindly provided by Dr. G. Suske . Plasmid constructs used in this study were sequenced with ABI3730XL DNA sequencer at the genomics facility of Macrogen (Seoul, South Korea).
Transient transfection and luciferase assay
Transient transfections with the pGL3-T2P plasmids, NF-κB-luc and IL-8-luc reporter constructs were carried out using the TransIT-LT1 transfection reagent (Panvera, Madison, WI, USA) as previously described . In brief, subconfluent cells in 12-well plates were transfected with 0.4 μg reporter plasmid and 10 ng of the Renilla luciferase vector (phRG-TK; Promega). The empty pcDNA3.1 vector was used as a control for the TLR2 and SP1/3 transfection experiments. After 48 hrs, the transfected and control cells were harvested and luciferase activity was measured by the Dual-Luciferase Reporter Assay system (Promega) in a luminometer. Relative luciferase activity is presented as % activity of each control described in each figure legend. Values shown are the mean ± S.E.M. (n = 3).
Semi-quantitative RT-PCR analysis
Total RNA from human epithelial cells was isolated using ISOGEN (NIPPONGENE, Tokyo, Japan) according to the manufacturer's instruction. Semi-quantitative RT-PCR was carried out using the RNA-PCR Kit (TaKaRa, Tokyo, Japan). PCR amplifications of TLR2 and GAPDH was performed as previously described . The amplification conditions for SP1 and SP3 are as follows: 94°C for 60 s, 60°C for 60 s, and 72°C for 60 s (for 24 cycles). The oligonucleotide primers used in the PCR amplifications are as indicated (Table 1). Quantitative analyses were performed by using Image Gauge (FUJI FILM, Tokyo, Japan).
SP1 and SP3 protein expression was assayed as previously described . Equal amounts of nuclear protein extract (30 μg) were fractionated by 8% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). The membrane was blocked with PBS-T (PBS, 0.1% (v/v) Tween 20) and 5% nonfat milk. Detection of SP1 and SP3 with anti-SP1 and anti-SP3 rabbit polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was carried as described . Actin expression was determined using a goat anti-actin polyclonal antibody (Santa Cruz Biotechnology) as the internal control.
Electrophoresis mobility shift assay (EMSA)
EMSA was performed as described . Briefly, nuclear extracts (7.5 μg) from 16HBE14o- and CFBE41o- cells were incubated with [γ-32P]-labeled oligonucleotides representing TLR2 promoter region -64/-31 with or without the indicated excess of unlabeled competitors. The DNA-protein complexes were resolved in 4.5% polyacrylamide gels. The SP1 antibody (4 μg) was used for supershift assays, and the migrating bands were visualized autoradiographically using BAS-2000 (Fuji film, Japan). The oligonucleotides used in the EMSA are as indicated (Table 1).
Statistical analysis of luciferase activity was performed by one-way ANOVA with either Tukey-Kramer or Dunnett's multiple comparison test (JMP software, SAS Institute, NC, USA) as indicated in each figure legends. Statistical analysis of methylation (Tables 2 and 3) was performed by the Fischer's exact test to assign p values.
This work was supported by funds from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, the Takeda Science Foundation, and in part by Global COE Program (Cell Fate Regulation Research and Education Unit), MEXT, Japan and grants from the Cystic Fibrosis Foundation and Pennsylvania Cystic Fibrosis, Inc (DCG). We confirmed that no conflict of financial interest exists in this work.
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