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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online February 14, 2006 as doi:10.1096/fj.05-4934fje. |
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* Department of Molecular Medicine, Graduate School of Pharmaceutical Science, Kumamoto University, Kumamoto, Japan;
Gonda Department of Cell and Molecular Biology, House Ear Institute, University of Southern California, Los Angeles, California, USA;
California Pacific Medical Center Research Institute, San Francisco, California, USA;
Department of Laboratory Medicine, University of California, San Francisco, San Francisco, California, USA;
|| Department of Medicine, University of Vermont, Burlington, Vermont, USA; and
¶ Department of Microbiology and Immunology, Tohoku University Graduate School of Dentistry, Sendai, Japan
1Correspondence: Department of Molecular Medicine, Graduate School of Pharmaceutical Science, Kumamoto University, Kumamoto 862-0973, Japan. E-mail: hirokai{at}gpo.kumamoto-u.ac.jp
SPECIFIC AIMS
The clinical course of cystic fibrosis (CF) is characterized by recurrent pulmonary infections and chronic inflammation. However, the molecular mechanisms underlying the enhanced inflammation to bacterial infections remain unclear. Increased proinflammatory responses toward Toll-like receptor-2 (TLR2) ligands in CF airway epithelial cells have been proposed as contributing to this enhanced chronic inflammation. The aims of this study are to confirm that aberrant regulation of TLR2 expression occurs in CF bronchial epithelial cells and to clarify the molecular mechanisms involved in dysregulated expression of TLR2 gene in CF bronchial epithelial cells.
PRINCIPAL FINDINGS
1. Increased expression of TLR2 gene and enhanced inflammatory response against TLR2 ligand in human CF bronchial epithelial cells
First, we sought to compare the expression level of Toll-like receptor-2 gene in non-CF (16HBE14o–) and CF (CFBE41o–) bronchial epithelial cells. Semiquantitative RT-PCR analysis using TLR2 specific primers was performed. The studies showed higher levels of TLR2 expression in CFBE41o– cells than in 16HBE14o– cells. Although detectable, mitigated expression of TLR2 gene was also observed in non-CF, 16HBE14o– cells (Fig. 1
A). Similar results were observed by real-time quantitative RT-PCR analysis. Consistent with the TLR2 expression in these cells, induction of endogenous IL-8 expression by Staphylococcus aureus peptidoglycan (PGN) was greatly enhanced in CFBE41o– cells, but not in 16HBE14o– cells.
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2. 5-Azacytidine increases TLR2 expression and demethylation in TLR2 proximal promoter
To understand the molecular mechanisms underlying increased expression of TLR2 in CFBE41o– cells, the studies undertaken focused on epigenetic gene regulation, since it has been shown that the TLR2 promoter has numerous potential DNA methylation sites (CpG motifs). To investigate the potential role of CpG methylation in the CF-restricted up-regulation of TLR2, we first analyzed the effect of 5-azacytidine (5-AC), a compound known to inhibit DNA methylation, on TLR2 expression in 16HBE14o– and CFBE41o– cells. 5-AC dose-dependently (1–10 µM) up-regulated TLR2 gene expression in 16HBE14o– cells to levels similar to that observed in CFBE41o– cells. To verify that 5-AC directly inhibited the methylation of the TLR2 promoter in 16HBE14o– cells, the methylation status of 20 CpGs within the proximal TLR2 promoter region up to –840 bp from the translation start site (designated as +1) was analyzed by bisulfite sequencing. The frequency of methylation and the average percent of methylation at each CpG site within all CpG were greatly reduced by the treatment of 16HBE14o– cells with 5-AC. These results clearly showed that inhibition of CpG methylation within the TLR2 promoter activates TLR2 gene expression in 16HBE14o– cells.
3. Promoter hypomethylation of TLR2 promoter is associated with TLR2 expression in other epithelial cells
To determine whether promoter methylation status of TLR2 gene is generally associated with TLR2 gene expression, expression of TLR2 gene in several non-CF and CF epithelial cell lines were analyzed by RT-PCR. This analysis showed increased expression of TLR2 mRNA in other CF epithelial cells (CFTE29o–, CFPAC-1) when compared with non-CF epithelial cells (16HBE14o–, A549, HeLa) (Fig. 1A
). To determine whether increased expression of TLR2 gene is associated with promoter hypomethylation of TLR2 gene in these epithelial cells, the methylation status of TLR2 promoter was analyzed. In CF epithelial cells, the frequency of methylated CpG in most of the CpG sites was lower than that in non-CF epithelial cells (Fig. 1B
). The average percent of methylation at each CpG site within all TLR2 proximal promoter CpGs was dramatically reduced in CF epithelial cells (Fig. 1C
), consistent with the increased expression of TLR2 mRNA in these cells.
4. Expression of WT-CFTR in CFBE41o– cells down-regulates TLR2 expression and modulates PGN response by altering TLR2 promoter methylation
To assess whether epigenetic down-regulation of TLR2 gene expression in CF epithelial cells could be modulated by WT-CFTR, CFBE41o– cells stably expressing WT-CFTR (CFBE41o–/WT-CFTR cells) were generated. CFBE41o– cells expressing 
F508-CFTR (CFBE41o–/F508-CFTR cells) were also generated as a control for expression vector-induced CFTR expression. As shown in Fig. 2
A, expression of the WT-CFTR, but not F508-CFTR, in CFBE41o– cells resulted in down-regulation of TLR2 expression. Similar results were also obtained by real-time PCR analysis (Fig. 2B
). Bisulfite sequencing analysis revealed that, in CFBE41o–/WT-CFTR cells, the average percent of methylation at each CpG site within the TLR2 proximal promoter was significantly increased in CFBE41o–/WT-CFTR cells to levels similar to that observed in the non-CF 16HBE14o– cells (Fig. 2C
). Consistent with the modulation of TLR2 expression, induction of endogenous IL-8 mRNA expression by PGN was greatly inhibited in CFBE41o–/WT-CFTR cells, but not in CFBE41o–/F508-CFTR cells (Fig. 2D
). The results from these isogenic cell line systems further support the hypothesis that expression of TLR2 is regulated in bronchial epithelial cells via a CFTR-dependent epigenetic mechanism.
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CONCLUSIONS AND SIGNIFICANCE
CF is one of the most common lethal genetic disorders among Caucasians and affects an estimated 30,000 persons in the U.S. CF is characterized by chronic lung infections with bacteria, intense neutrophil-dominated airway inflammation, and progressive lung disease, which is the major cause of morbidity and mortality. So that identification of CF-specific inflammatory pathways can be an optimal target for CF therapy. Although much work has been done on understanding the mechanisms responsible for the enhanced inflammation to bacterial infections, they remain unclear. Aberrant regulation of the immune response or hyperesponsiveness to the bacterial ligands has been proposed as a cause of the enhanced susceptibility to this chronic inflammation. In this report, we confirmed that increased expression of TLR2 occur in bronchial epithelial cells from CF. This finding will bring one of the critical answers to the question of why chronic inflammation occur in CF patients. Our results may have important implication for the development of therapeutic regimens directed at the pharmacological regulation of TLR2.
Our study also shows the first solid evidence that the TLR2 promoter is highly methylated in non-CF epithelial cells and demethylated in CF epithelial cells. We also demonstrated that the methylation of CpG sites in the TLR2 promoter were increased after introduction of WT-CFTR into CFBE41o– cells, thereby suggesting that a functional CFTR has the ability to modulate the methylation status of TLR2 gene and that this altered methylation might be responsible for CF-associated up-regulation of TLR2. To date, no one has shown the involvement of aberrant CpG methylation in CF pathogenesis; therefore, this present study provides a novel insight into a mechanism underlying CF pathogenesis.
We conclude that the TLR2 gene expression and the cellular response to PGN were strongly enhanced in human CF bronchial epithelial cells. As presented in Fig. 3
, promoter hypomethylation of TLR2 gene is responsible for increased expression of TLR2 gene and enhanced inflammatory response against PGN in human CF bronchial epithelial cells. We also showed that CFTR existence is important to regulate TLR2 gene expression in human bronchial epithelial cells. How CFTR regulates CpG methylation status in non-CF and CF epithelial cells remains an open question. However, it is reasonable to think that CFTR affects the function of DNA methyl transferase family members and/or DNA demethylase, MBD2 since many papers suggested the multiple function of CFTR to control a number of physiological phenomena by interacting with other proteins with a variety of function.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4934fje;
This article has been cited by other articles:
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